Pcsk9 targeting oligonucleotides for treating hypercholesterolemia and related conditions

ABSTRACT

This disclosure relates to oligonucleotides, compositions and methods useful for reducing PCSK9 expression, particularly in hepatocytes. Disclosed oligonucleotides for the reduction of PCSK9 expression may be double-stranded or single-stranded, and may be modified for improved characteristics such as stronger resistance to nucleases and lower immunogenicity. Disclosed oligonucleotides for the reduction of PCSK9 expression may also include targeting ligands to target a particular cell or organ, such as the hepatocytes of the liver, and may be used to treat hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 17/048,846, filed Oct. 19, 2020, which is a National Stage application, filed under 35 U.S.C. § 371, of International Application No. PCT/US2019/025253, filed Apr. 1, 2019, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/659,693, filed Apr. 18, 2018, and U.S. Provisional Application No. 62/820,558, filed Mar. 19, 2019, the entire contents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present application relates to oligonucleotides and uses thereof, particularly uses relating to the treatment of hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 400930-020USD1-195261.txt created on Nov. 30, 2022 which is 257 kilobytes in size. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Cholesterol is one of three major classes of lipids manufactured by animal cells and used to construct cell membranes. Cholesterol is water insoluble and transported in the blood plasma within protein particles (lipoproteins). Any lipoprotein (e.g., very low density lipoprotein (VLDL), low density lipoprotein (LDL), intermediate density lipoprotein (IDL) and high density lipoprotein (HDL)) may carry cholesterol, but elevated levels of non-HDL cholesterol (most particularly LDL-cholesterol) are associated with an increased risk of atherosclerosis and coronary heart disease (e.g., coronary artery disease). This type of elevated cholesterol is known as hypercholesterolemia. Hypercholesterolemia can lead to the deposition of plaques on artery walls, known as atherosclerosis. Proprotein convertase subtilisin/kexin-9 (also known as PCSK9) is a serine protease that indirectly regulates plasma LDL cholesterol levels by controlling both hepatic and extrahepatic LDL receptor (LDLR) expression at the plasma membrane. Decreased expression of the PCSK9 protein increases expression of the LDLR receptor, thereby decreasing plasma LDL cholesterol and the resultant hypercholesterolemia and/or atherosclerosis as well as complications arising from the same.

BRIEF SUMMARY OF THE INVENTION

Aspects of the disclosure relate to oligonucleotides and related methods for treating hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof in a subject. In some embodiments, potent RNAi oligonucleotides have been developed for selectively inhibiting PCSK9 expression in a subject. In some embodiments, the RNAi oligonucleotides are useful for reducing PCSK9 activity, and thereby decreasing or preventing hypercholesterolemia (high levels of low density lipoprotein (LDL)-cholesterol), atherosclerosis, and/or one or more symptoms or complications thereof. In some embodiments, key regions of PCSK9 mRNA (referred to as hotspots) have been identified herein that are particularly amenable to targeting using such oligonucleotide-based approaches (See Example 1).

One aspect of the present disclosure provides oligonucleotides for reducing expression of PCSK9. In some embodiments, the oligonucleotides comprise an antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, 1193-1232, 1257-1265, or 1269-1271. In some embodiments, the oligonucleotides further comprise a sense strand that comprises a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, 1153-1192, 1248-1256, or 1266-1268. In some embodiments, the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, 1193-1232, 1257-1265, or 1269-1271. In some embodiments, the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, 1153-1192, 1248-1256, or 1266-1268. One aspect of the present disclosure provides oligonucleotides for reducing expression of PCSK9, in which the oligonucleotides comprise an antisense strand of 15 to 30 nucleotides in length. In some embodiments, the antisense strand has a region of complementarity to a target sequence of PCSK9 as set forth in any one of SEQ ID NOs: 1233-1244. In some embodiments, the region of complementarity is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides in length. In some embodiments, the region of complementarity is fully complementary to the target sequence of PCSK9. In some embodiments, the region of complementarity is at least 19 contiguous nucleotides in length.

In some embodiments, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, or 1153-1192. In some embodiments, the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, or 1153-1192. In some embodiments, the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, or 1193-1232. In some embodiments, the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, or 1193-1232.

In some embodiments, the antisense strand is 19 to 27 nucleotides in length. In some embodiments, the antisense strand is 21 to 27 nucleotides in length. In some embodiments, the oligonucleotide further comprises a sense strand of 15 to 40 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand. In some embodiments, the sense strand is 19 to 40 nucleotides in length. In some embodiments, the duplex region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the antisense strand and sense strand form a duplex region of 25 nucleotides in length.

In some embodiments, an oligonucleotide comprises an antisense strand and a sense strand that are each in a range of 21 to 23 nucleotides in length. In some embodiments, an oligonucleotide comprises a duplex structure in a range of 19 to 21 nucleotides in length. In some embodiments, an oligonucleotide comprises a 3′-overhang sequence of one or more nucleotides in length, in which the 3′-overhang sequence is present on the antisense strand, the sense strand, or the antisense strand and sense strand. In some embodiments, an oligonucleotide further comprises a 3′-overhang sequence on the antisense strand of two nucleotides in length. In some embodiments, an oligonucleotide comprises a 3′-overhang sequence of two nucleotides in length, in which the 3′-overhang sequence is present on the antisense strand, and in which the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and antisense strand form a duplex of 21 nucleotides in length.

Another aspect of the present disclosure provides an oligonucleotide for reducing expression of PCSK9, the oligonucleotide comprising an antisense strand and a sense strand, in which the antisense strand is 21 to 27 nucleotides in length and has a region of complementarity to PCSK9, in which the sense strand comprises at its 3′-end a stem-loop set forth as: S₁-L-S₂, in which S₁ is complementary to S₂, and in which L forms a loop between S₁ and S₂ of 3 to 5 nucleotides in length, and in which the antisense strand and the sense strand form a duplex structure of at least 19 nucleotides in length but are not covalently linked. In some embodiments, the sense strand comprises at its 3′-end a stem-loop set forth as: S₁-L-S₂, in which S₁ is complementary to S₂, and in which L forms a loop between S₁ and S₂ of 3 to 5 nucleotides in length. In some embodiments, the region of complementarity is fully complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides of PCSK9 mRNA. In some embodiments, L is a tetraloop. In some embodiments, L is 4 nucleotides in length. In some embodiments, L comprises a sequence set forth as GAAA.

In some embodiments, an oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2′-modification. In some embodiments, the 2′-modification is a modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. In some embodiments, all of the nucleotides of an oligonucleotide are modified.

In some embodiments, an oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog. In some embodiments, the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

In some embodiments, at least one nucleotide of an oligonucleotide is conjugated to one or more targeting ligands. In some embodiments, each targeting ligand comprises a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid. In some embodiments, each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety. In some embodiments, up to 4 nucleotides of L of the stem-loop are each conjugated to a monovalent GalNAc moiety. In other embodiments, a bi-valent, tri-valent, or tetravalent GalNac moiety is conjugated to a single nucleotide, e.g., of the nucleotides of L of a stem loop. In some embodiments, the targeting ligand comprises an aptamer.

Another aspect of the present disclosure provides a composition comprising an oligonucleotide of the present disclosure and an excipient. Another aspect of the present disclosure provides a method comprising administering a composition of the present disclosure to a subject. In some embodiments, the method results in a decrease in level or severity of, or results in prevention of, hypercholesterolemia (high levels of low density lipoprotein (LDL)-cholesterol), atherosclerosis, coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke (i.e., death of brain cells resulting from insufficient blood and oxygen flow to the brain), feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, loss of consciousness, peripheral artery disease, and/or kidney problems (e.g., chronic kidney disease). Another aspect of the present disclosure provides a method for treating hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof.

Another aspect of the present disclosure provides an oligonucleotide for reducing expression of PCSK9, the oligonucleotide comprising a sense strand of 15 to 40 nucleotides in length and an antisense strand of 15 to 30 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand, in which the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, 1153-1192, 1248-1256, or 1266-1268 and the antisense strand comprises a complementary sequence selected from SEQ ID NOs: 454-906, 1030-1152, 1193-1232, 1257-1265, or 1269-1271.

In some embodiments, the oligonucleotide comprises a pair of sense and antisense strands selected from a row of the table set forth in Table 4.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein.

FIGS. 1A and 1B are graphs showing the percentage of PCSK9 mRNA remaining after a screen of 576 PCSK9 oligonucleotides in Huh-7 cells. The nucleotide position in NM_174936.3 that corresponds to the 3′ end of the sense strand of each siRNA is indicated on the x axis.

FIGS. 2A-2D are a set of graphs showing the percentage of mRNA remaining after PCSK9 oligonucleotide screening of 96 PCSK9 oligonucleotides at two different concentrations (0.1 nM and 1 nM) in Huh-7 cells. The H number on the X-axis indicates the position in the PCSK9 mRNA mapping to the 5′ end of the antisense strand of the oligonucleotides.

FIG. 3 is a schematic showing a non-limiting example of a double-stranded oligonucleotide with a nicked tetraloop structure that has been conjugated to four GalNAc moieties (diamond shapes).

FIG. 4 is a graph showing the results of screening in a mouse hydrodynamic injection (HDI) model using PCSK9 tetraloop conjugates of 12 different base sequences with a single modification pattern. PBS, shown on the far left, was used as a control.

FIGS. 5A-5C are graphs showing the results of screening in Huh-7 cells (FIG. 5A) and in a mouse HDI model (FIGS. 5B and 5C) using PCSK9 oligonucleotides of different base sequences. FIG. 5A is a graph showing the percentage of PCSK9 mRNA remaining after screening of 40 nicked-tetraloop structures. The same modification pattern was used, and the oligonucleotides were tested at two different concentrations (0.03 nM and 0.1 nM; labeled as “Phase T2” in FIG. 5A). FIG. 5B shows a human-specific PCSK9 tetraloop conjugate screen in the mouse HDI model at a 2 mg/kg subcutaneous dose using three different modification patterns. FIG. 5C shows the same test as described in FIG. 5B, except at a 1 mg/kg subcutaneous dose (except for the control, which was dosed at both 1 and 2 mg/kg). Two different modification patterns were used. PBS was used as a control and is shown to the left.

FIGS. 6A and 6B are graphs showing the results of screening in a mouse hydrodynamic injection (HDI) model using three different PCSK9 tetraloop conjugates with varied modification patterns at three different concentrations. PBS, shown on the far left, was used as a control.

FIGS. 7A-7D are graphs showing an in vivo activity evaluation of PCSK9 oligonucleotides in a tetraloop conjugate in non-human primates. Candidate sequences were tested with different modifications. FIG. 7A shows the analysis of PCSK9 remaining and LDL-C lowering using a candidate PCSK9 tetraloop conjugate with two different modification patterns. The ability of the oligonucleotide to lower plasma PCSK9 through Day 30 (FIG. 7B) and through Day 90 (FIG. 7C) was measured using a PCSK9 ELISA. Serum levels of LDL were also measured, as shown in FIG. 7D.

DETAILED DESCRIPTION OF THE INVENTION

According to some aspects, the disclosure provides oligonucleotides targeting PCSK9 mRNA that are effective for reducing PCSK9 expression in cells, particularly liver cells (e.g., hepatocytes) for the treatment of hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof. Accordingly, in related aspects, the disclosure provides methods of treating hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof that involve selectively reducing PCSK9 gene expression in liver. In certain embodiments, PCSK9 targeting oligonucleotides provided herein are designed for delivery to selected cells of target tissues (e.g., liver hepatocytes) to treat hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof in a subject.

Further aspects of the disclosure, including a description of defined terms, are provided below.

I. Definitions

Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Administering: As used herein, the terms “administering” or “administration” means to provide a substance (e.g., an oligonucleotide) to a subject in a manner that is pharmacologically useful (e.g., to treat a condition in the subject).

Asialoglycoprotein receptor (ASGPR): As used herein, the term “Asialoglycoprotein receptor” or “ASGPR” refers to a bipartite C-type lectin formed by a major 48 kDa (ASGPR-1) and minor 40 kDa subunit (ASGPR-2). ASGPR is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins).

Atherosclerosis: As used herein, the term “atherosclerosis” refers to a disease involving a narrowing of arteries (e.g., coronary, carotid, peripheral, and/or renal arteries) typically due to the buildup of plaques (made from fat, cholesterol, calcium, and other substances). In some embodiments, narrowing of the coronary arteries may produce symptoms such as angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, and/or palpitations. In some embodiments, narrowing of the carotid arteries may result in a stroke (i.e., death of brain cells resulting from insufficient blood and oxygen flow to the brain) and/or may produce symptoms such as feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, and/or loss of consciousness. In some embodiments, narrowing of the peripheral arteries may result in numbness or pain within the arms or legs. In some embodiments, narrowing of the renal arteries (resulting in decreased kidney blood flow) may result in chronic kidney disease. Complications of atherosclerosis may include coronary artery disease, stroke, peripheral artery disease, and kidney problems (e.g., chronic kidney disease).

Complementary: As used herein, the term “complementary” refers to a structural relationship between nucleotides (e.g., on two nucleotides on opposing nucleic acids or on opposing regions of a single nucleic acid strand) that permits the nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. In some embodiments, complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. In some embodiments, two nucleic acids may have nucleotide sequences that are complementary to each other so as to form regions of complementarity, as described herein.

Deoxyribonucleotide: As used herein, the term “deoxyribonucleotide” refers to a nucleotide having a hydrogen at the 2′ position of its pentose sugar as compared with a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than at the 2′ position, including modifications or substitutions in or of the sugar, phosphate group or base.

Double-stranded oligonucleotide: As used herein, the term “double-stranded oligonucleotide” refers to an oligonucleotide that is substantially in a duplex form. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of covalently separate nucleic acid strands. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of nucleic acid strands that are covalently linked. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed from a single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are fully duplexed with one another. However, in some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are partially duplexed, e.g., having overhangs at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and thus, may have one or more mismatches, which may include internal mismatches or end mismatches.

Duplex: As used herein, the term “duplex,” in reference to nucleic acids (e.g., oligonucleotides), refers to a structure formed through complementary base-pairing of two antiparallel sequences of nucleotides.

Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect.

Hepatocyte: As used herein, the term “hepatocyte” or “hepatocytes” refers to cells of the parenchymal tissues of the liver. These cells make up approximately 70-85% of the liver's mass and manufacture serum albumin, fibrinogen, and the prothrombin group of clotting factors (except for Factors 3 and 4). Markers for hepatocyte lineage cells may include, but are not limited to: transthyretin (Ttr), glutamine synthetase (Glul), hepatocyte nuclear factor 1a (Hnfla), and hepatocyte nuclear factor 4a (Hnf4a). Markers for mature hepatocytes may include, but are not limited to: cytochrome P450 (Cyp3a11), fumarylacetoacetate hydrolase (Fah), glucose 6-phosphate (G6p), albumin (Alb), and OC2-2F8. See, e.g., Huch et al., (2013), Nature, 494(7436): 247-250, the contents of which relating to hepatocyte markers is incorporated herein by reference.

Hypercholesterolemia: As used herein, the term “hypercholesterolemia” refers to the presence of high levels of cholesterol (e.g., low density lipoprotein (LDL)-cholesterol) in the blood. Cholesterol is one of three major classes of lipids manufactured by animal cells and used to construct cell membranes. Cholesterol is water insoluble and transported in the blood plasma within protein particles (lipoproteins). Any lipoprotein (e.g., very low density lipoprotein (VLDL), low density lipoprotein (LDL), intermediate density lipoprotein (IDL) and high density lipoprotein (HDL)) may carry cholesterol, but elevated levels of non-HDL cholesterol (most particularly LDL-cholesterol) are associated with an increased risk of atherosclerosis and coronary heart disease (e.g., coronary artery disease).

Loop: As used herein, the term “loop” refers to an unpaired region of a nucleic acid (e.g., oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to one another, such that under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cells), the two antiparallel regions, which flank the unpaired region, hybridize to form a duplex (referred to as a “stem”).

Modified Internucleotide Linkage: As used herein, the term “modified internucleotide linkage” refers to an internucleotide linkage having one or more chemical modifications compared with a reference internucleotide linkage comprising a phosphodiester bond. In some embodiments, a modified nucleotide is a non-naturally occurring linkage. Typically, a modified internucleotide linkage confers one or more desirable properties to a nucleic acid in which the modified internucleotide linkage is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.

Modified Nucleotide: As used herein, the term “modified nucleotide” refers to a nucleotide having one or more chemical modifications compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. In some embodiments, a modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, a modified nucleotide has one or more chemical modifications in its sugar, nucleobase and/or phosphate group. In some embodiments, a modified nucleotide has one or more chemical moieties conjugated to a corresponding reference nucleotide. Typically, a modified nucleotide confers one or more desirable properties to a nucleic acid in which the modified nucleotide is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc. In certain embodiments, a modified nucleotide comprises a 2′-O-methyl or a 2′-F substitution at the 2′ position of the ribose ring.

Nicked Tetraloop Structure: A “nicked tetraloop structure” is a structure of a RNAi oligonucleotide characterized by the presence of separate sense (passenger) and antisense (guide) strands, in which the sense strand has a region of complementarity to the antisense strand such that the two strands form a duplex, and in which at least one of the strands, generally the sense strand, extends from the duplex in which the extension contains a tetraloop and two self-complementary sequences forming a stem region adjacent to the tetraloop, in which the tetraloop is configured to stabilize the adjacent stem region formed by the self-complementary sequences of the at least one strand.

Oligonucleotide: As used herein, the term “oligonucleotide” refers to a short nucleic acid, e.g., of less than 100 nucleotides in length. An oligonucleotide can comprise ribonucleotides, deoxyribonucleotides, and/or modified nucleotides including, for example, modified ribonucleotides. An oligonucleotide may be single-stranded or double-stranded. An oligonucleotide may or may not have duplex regions. As a set of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA, or single-stranded siRNA. In some embodiments, a double-stranded oligonucleotide is an RNAi oligonucleotide.

Overhang: As used herein, the term “overhang” refers to terminal non-base-pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of a complementary strand with which the one strand or region forms a duplex. In some embodiments, an overhang comprises one or more unpaired nucleotides extending from a duplex region at the 5′ terminus or 3′ terminus of a double-stranded oligonucleotide. In certain embodiments, the overhang is a 3′ or 5′ overhang on the antisense strand or sense strand of a double-stranded oligonucleotide.

Phosphate analog: As used herein, the term “phosphate analog” refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5′ terminal nucleotide of an oligonucleotide in place of a 5′-phosphate, which is often susceptible to enzymatic removal. In some embodiments, a 5′ phosphate analog contains a phosphatase-resistant linkage. Examples of phosphate analogs include 5′ phosphonates, such as 5′ methylenephosphonate (5′-MP) and 5′-(E)-vinylphosphonate (5′-VP). In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”) at a 5′-terminal nucleotide. An example of a 4′-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. See, for example, International Patent Application PCT/US2017/049909, filed on Sep. 1, 2017, U.S. Provisional Application Nos. 62/383,207, filed on Sep. 2, 2016, and 62/393,401, filed on Sep. 12, 2016, the contents of each of which relating to phosphate analogs are incorporated herein by reference. Other modifications have been developed for the 5′ end of oligonucleotides (see, e.g., WO 2011/133871; U.S. Pat. No. 8,927,513; and Prakash et al. (2015), Nucleic Acids Res., 43(6):2993-3011, the contents of each of which relating to phosphate analogs are incorporated herein by reference).

Proprotein convertase subtilisin/kexin-9 (PCSK9): As used herein, the term “proprotein convertase subtilisin/kexin-9” (also known as PCSK9, NARC-1, neural apoptosis regulated convertase 1, HCHOLA3, and hypercholesterolemia, autosomal dominant 3) refers to the gene encoding PCSK9 protein.

Reduced expression: As used herein, the term “reduced expression” of a gene refers to a decrease in the amount of RNA transcript or protein encoded by the gene and/or a decrease in the amount of activity of the gene in a cell or subject, as compared to an appropriate reference cell or subject. For example, the act of treating a cell with a double-stranded oligonucleotide (e.g., one having an antisense strand that is complementary to PCSK9 mRNA sequence) may result in a decrease in the amount of RNA transcript, protein and/or enzymatic activity (e.g., encoded by the PCSK9 gene) compared to a cell that is not treated with the double-stranded oligonucleotide. Similarly, “reducing expression” as used herein refers to an act that results in reduced expression of a gene (e.g., PCSK9).

Region of Complementarity: As used herein, the term “region of complementarity” refers to a sequence of nucleotides of a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides (e.g., a target nucleotide sequence within an mRNA) to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions, e.g., in a phosphate buffer, in a cell, etc. A region of complementarity may be fully complementary to a nucleotide sequence (e.g., a target nucleotide sequence present within an mRNA or portion thereof). For example, a region of complementary that is fully complementary to a nucleotide sequence present in an mRNA has a contiguous sequence of nucleotides that is complementary, without any mismatches or gaps, to a corresponding sequence in the mRNA. Alternatively, a region of complementarity may be partially complementary to a nucleotide sequence (e.g., a nucleotide sequence present in an mRNA or portion thereof). For example, a region of complementary that is partially complementary to a nucleotide sequence present in an mRNA has a contiguous sequence of nucleotides that is complementary to a corresponding sequence in the mRNA but that contains one or more mismatches or gaps (e.g., 1, 2, 3, or more mismatches or gaps) compared with the corresponding sequence in the mRNA, provided that the region of complementarity remains capable of hybridizing with the mRNA under appropriate hybridization conditions.

Ribonucleotide: As used herein, the term “ribonucleotide” refers to a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2′ position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than at the 2′ position, including modifications or substitutions in or of the ribose, phosphate group or base.

RNAi Oligonucleotide: As used herein, the term “RNAi oligonucleotide” refers to either (a) a double stranded oligonucleotide having a sense strand (passenger) and antisense strand (guide), in which the antisense strand or part of the antisense strand is used by the Argonaute 2 (Ago2) endonuclease in the cleavage of a target mRNA or (b) a single stranded oligonucleotide having a single antisense strand, where that antisense strand (or part of that antisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA.

Strand: As used herein, the term “strand” refers to a single contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages, phosphorothioate linkages). In some embodiments, a strand has two free ends, e.g., a 5′-end and a 3′-end.

Subject: As used herein, the term “subject” means any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or non-human primate. The terms “individual” or “patient” may be used interchangeably with “subject.”

Synthetic: As used herein, the term “synthetic” refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid state nucleic acid synthesizer)) or that is otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

Targeting ligand: As used herein, the term “targeting ligand” refers to a molecule (e.g., a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and that is conjugatable to another substance for purposes of targeting the other substance to the tissue or cell of interest. For example, in some embodiments, a targeting ligand may be conjugated to an oligonucleotide for purposes of targeting the oligonucleotide to a specific tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Accordingly, in some embodiments, a targeting ligand when conjugated to an oligonucleotide facilitates delivery of the oligonucleotide into a particular cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of the complex comprising the oligonucleotide, targeting ligand, and receptor. In some embodiments, a targeting ligand is conjugated to an oligonucleotide via a linker that is cleaved following or during cellular internalization such that the oligonucleotide is released from the targeting ligand in the cell.

Tetraloop: As used herein, the term “tetraloop” refers to a loop that increases stability of an adjacent duplex formed by hybridization of flanking sequences of nucleotides. The increase in stability is detectable as an increase in melting temperature (T_(m)) of an adjacent stem duplex that is higher than the T_(m) of the adjacent stem duplex expected, on average, from a set of loops of comparable length consisting of randomly selected sequences of nucleotides. For example, a tetraloop can confer a melting temperature of at least 50° C., at least 55° C., at least 56° C., at least 58° C., at least 60° C., at least 65° C., or at least 75° C. in 10 mM NaHPO₄ to a hairpin comprising a duplex of at least 2 base pairs in length. In some embodiments, a tetraloop may stabilize a base pair in an adjacent stem duplex by stacking interactions. In addition, interactions among the nucleotides in a tetraloop include, but are not limited to non-Watson-Crick base-pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug. 16; 346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016):191-4). In some embodiments, a tetraloop comprises or consists of 3 to 6 nucleotides, and is typically 4 to 5 nucleotides. In certain embodiments, a tetraloop comprises or consists of three, four, five, or six nucleotides, which may or may not be modified (e.g., which may or may not be conjugated to a targeting moiety). In one embodiment, a tetraloop consists of four nucleotides. Any nucleotide may be used in the tetraloop and standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden (1985) Nucl. Acids Res. 13: 3021-3030. For example, the letter “N” may be used to mean that any base may be in that position, the letter “R” may be used to show that A (adenine) or G (guanine) may be in that position, and “B” may be used to show that C (cytosine), G (guanine), or T (thymine) may be in that position. Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al., Proc Natl Acad Sci USA. 1990 November; 87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov. 11; 19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA)), the d(GNRA) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, for example: Nakano et al. Biochemistry, 41 (48), 14281-14292, 2002. SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000), which are incorporated by reference herein for their relevant disclosures. In some embodiments, the tetraloop is contained within a nicked tetraloop structure.

Treat: As used herein, the term “treat” refers to the act of providing care to a subject in need thereof, e.g., through the administration a therapeutic agent (e.g., an oligonucleotide) to the subject, for purposes of improving the health and/or well-being of the subject with respect to an existing condition (e.g., a disease, disorder) or to prevent or decrease the likelihood of the occurrence of a condition. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom or contributing factor of a condition (e.g., disease, disorder) experienced by a subject.

II. Oligonucleotide-Based Inhibitors

i. PCSK9 Targeting Oligonucleotides

Potent oligonucleotides have been identified herein through examination of the PCSK9 mRNA, including mRNAs of different species (human and Rhesus macaque, (see, e.g., Example 1)) and in vitro and in vivo testing. Such oligonucleotides can be used to achieve therapeutic benefit for subjects with a hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof by reducing PCSK9 activity, and consequently, by decreasing or preventing hypercholesterolemia (high levels of low density lipoprotein (LDL)-cholesterol), atherosclerosis, coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke (i.e., death of brain cells resulting from insufficient blood and oxygen flow to the brain), feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, loss of consciousness, peripheral artery disease, and/or kidney problems (e.g., chronic kidney disease). For example, potent RNAi oligonucleotides are provided herein that have a sense strand comprising, or consisting of, a sequence as set forth in any one of SEQ ID NO: 1-453, 907-1029, 1153-1192, 1248-1256, and 1266-1268 and an antisense strand comprising, or consisting of, a complementary sequence selected from SEQ ID NO: 454-906, 1030-1152, 1193-1232, 1257-1265, and 1269-1271, as is also arranged the table provided in Table 4 (e.g., a sense strand comprising a sequence as set forth in SEQ ID NO: 1 and an antisense strand comprising a sequence as set forth in SEQ ID NO: 454). The sequences can be put into multiple different structures (or formats), as described herein.

In some embodiments, it has been discovered that certain regions of PCSK9 mRNA are hotspots for targeting because they are more amenable than other regions to oligonucleotide-based inhibition. In some embodiments, a hotspot region of PCSK9 consists of a sequence as forth in any one of SEQ ID NOs: 1233-1244. These regions of PCSK9 mRNA may be targeted using oligonucleotides as discussed herein for purposes of inhibiting PCSK9 mRNA expression.

Accordingly, in some embodiments, oligonucleotides provided herein are designed so as to have regions of complementarity to PCSK9 mRNA (e.g., within a hotspot of PCSK9 mRNA) for purposes of targeting the mRNA in cells and inhibiting its expression. The region of complementarity is generally of a suitable length and base content to enable annealing of the oligonucleotide (or a strand thereof) to PCSK9 mRNA for purposes of inhibiting its expression.

In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is at least partially complementary to a sequence as set forth in any of SEQ ID NOs: 1-453 or 907-1029, which include certain sequences mapping to within hotspot regions of PCSK9 mRNA. In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is fully complementary to a sequence as set forth in any of SEQ ID NOs: 1-453 or 907-1029. In some embodiments, a region of complementarity of an oligonucleotide that is complementary to contiguous nucleotides of a sequence as set forth in any of SEQ ID NOs: 1-453 or 907-1029 spans the entire length of an antisense strand. In some embodiments, a region of complementarity of an oligonucleotide that is complementary to contiguous nucleotides of a sequence as set forth in any one of any of SEQ ID NOs: 1-453 or 907-1029 spans a portion of the entire length of an antisense strand (e.g., all but two nucleotides at the 3′ end of the antisense strand). In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is at least partially (e.g., fully) complementary to a contiguous stretch of nucleotides spanning nucleotides 1-19 of a sequence as set forth in SEQ ID NOs: 1153-1192.

In some embodiments, the region of complementarity is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length. In some embodiments, an oligonucleotide provided herein has a region of complementarity to PCSK9 mRNA that is in the range of 12 to 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length. In some embodiments, an oligonucleotide provided herein has a region of complementarity to PCSK9 mRNA that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, a region of complementarity to PCSK9 mRNA may have one or more mismatches compared with a corresponding sequence of PCSK9 mRNA. A region of complementarity on an oligonucleotide may have up to 1, up to 2, up to 3, up to 4, etc. mismatches provided that it maintains the ability to form complementary base pairs with PCSK9 mRNA under appropriate hybridization conditions. Alternatively, a region of complementarity on an oligonucleotide may have no more than 1, no more than 2, no more than 3, or no more than 4 mismatches provided that it maintains the ability to form complementary base pairs with PCSK9 mRNA under appropriate hybridization conditions. In some embodiments, if there are more than one mismatches in a region of complementarity, they may be positioned consecutively (e.g., 2, 3, 4, or more in a row), or interspersed throughout the region of complementarity provided that the oligonucleotide maintains the ability to form complementary base pairs with PCSK9 mRNA under appropriate hybridization conditions.

Still, in some embodiments, double-stranded oligonucleotides provided herein comprise, of consist of, a sense strand having a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, 1153-1192, 1248-1256, and 1266-1268 and an antisense strand comprising a complementary sequence selected from SEQ ID NOs: 454-906, 1030-1152, 1193-1232, 1257-1265, and 1269-1271, as is arranged in the table provided in Table 4 (e.g., a sense strand comprising a sequence as set forth in SEQ ID NO: 1 and an antisense strand comprising a sequence as set forth in SEQ ID NO: 454).

ii. Oligonucleotide Structures

There are a variety of structures of oligonucleotides that are useful for targeting PCSK9 mRNA in the methods of the present disclosure, including RNAi, miRNA, etc. Any of the structures described herein or elsewhere may be used as a framework to incorporate or target a sequence described herein (e.g., a hotpot sequence of PCSK9 such as those illustrated in SEQ ID NOs: 1233-1244 or a sense or antisense strand that comprises or consists of a sequence as set forth as any of SEQ ID NOs: 1 to 453, 907-1029, and 1153-1192 or as set forth as any of SEQ ID NOs: 454-906, 1030-1152, and 1193-1232). Double-stranded oligonucleotides for targeting PCSK9 expression (e.g., via the RNAi pathway) generally have a sense strand and an antisense strand that form a duplex with one another. In some embodiments, the sense and antisense strands are not covalently linked. However, in some embodiments, the sense and antisense strands are covalently linked.

In some embodiments, double-stranded oligonucleotides for reducing PCSK9 expression engage RNA interference (RNAi). For example, RNAi oligonucleotides have been developed with each strand having sizes of 19-25 nucleotides with at least one 3′ overhang of 1 to 5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed that are processed by the Dicer enzyme to generate active RNAi products (see, e.g., U.S. Pat. No. 8,883,996). Further work produced extended double-stranded oligonucleotides where at least one end of at least one strand is extended beyond a duplex targeting region, including structures where one of the strands includes a thermodynamically-stabilizing tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, as well as WO2010033225, which are incorporated by reference herein for their disclosure of these oligonucleotides). Such structures may include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.

In some embodiments, sequences described herein can be incorporated into, or targeted using, oligonucleotides that comprise separate sense and antisense strands that are both in the range of 17 to 40 nucleotides in length. In some embodiments, oligonucleotides incorporating such sequences are provided that have a tetraloop structure within a 3′ extension of their sense strand, and two terminal overhang nucleotides at the 3′ end of the separate antisense strand. In some embodiments, the two terminal overhang nucleotides are GG. Typically, one or both of the two terminal GG nucleotides of the antisense strand is or are not complementary to the target.

In some embodiments, oligonucleotides incorporating such sequences are provided that have sense and antisense strands that are both in the range of 21 to 23 nucleotides in length. In some embodiments, a 3′ overhang is provided on the sense, antisense, or both sense and antisense strands that is 1 or 2 nucleotides in length. In some embodiments, an oligonucleotide has a guide strand of 23 nucleotides and a passenger strand of 21 nucleotides, in which the 3′-end of passenger strand and 5′-end of guide strand form a blunt end and where the guide strand has a two nucleotide 3′ overhang.

In some embodiments, oligonucleotides may be in the range of 21 to 23 nucleotides in length. In some embodiments, oligonucleotides may have an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3′ end of the sense and/or antisense strands. In some embodiments, oligonucleotides (e.g., siRNAs) may comprise a 21 nucleotide guide strand that is antisense to a target RNA and a complementary passenger strand, in which both strands anneal to form a 19-bp duplex and 2 nucleotide overhangs at either or both 3′ ends. See, for example, U.S. Pat. Nos. 9,012,138, 9,012,621, and 9,193,753, the contents of each of which are incorporated herein for their relevant disclosures.

In some embodiments, an oligonucleotide of the invention has a 36 nucleotide sense strand that comprises a region extending beyond the antisense-sense duplex, where the extension region has a stem-tetraloop structure where the stem is a six base pair duplex and where the tetraloop has four nucleotides. In certain of those embodiments, three or four of the tetraloop nucleotides are each conjugated to a monovalent GalNac ligand.

In some embodiments, an oligonucleotide of the invention comprises a 25 nucleotide sense strand and a 27 nucleotide antisense strand that when acted upon by a dicer enzyme results in an antisense strand that is incorporated into the mature RISC.

Other oligonucleotides designs for use with the compositions and methods disclosed herein include: 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., having 19 bp or shorter stems; see, e.g., Moore et al. Methods Mol. Biol. 2010; 629:141-158), blunt siRNAs (e.g., of 19 bps in length; see: e.g., Kraynack and Baker, RNA Vol. 12, p 163-176 (2006)), asymmetrical siRNAs (aiRNA; see, e.g., Sun et al., Nat. Biotechnol. 26, 1379-1382 (2008)), asymmetric shorter-duplex siRNA (see, e.g., Chang et al., Mol Ther. 2009 April; 17(4): 725-32), fork siRNAs (see, e.g., Hohjoh, FEBS Letters, Vol 557, issues 1-3; January 2004, p 193-198), single-stranded siRNAs (Elsner; Nature Biotechnology 30, 1063 (2012)), dumbbell-shaped circular siRNAs (see, e.g., Abe et al. J Am Chem Soc 129: 15108-15109 (2007)), and small internally segmented interfering RNA (sisiRNA; see, e.g., Bramsen et al., Nucleic Acids Res. 2007 September; 35(17): 5886-5897). Each of the foregoing references is incorporated by reference in its entirety for the related disclosures therein. Further non-limiting examples of an oligonucleotide structures that may be used in some embodiments to reduce or inhibit the expression of PCSK9 are microRNA (miRNA), short hairpin RNA (shRNA), and short siRNA (see, e.g., Hamilton et al., Embo J., 2002, 21(17): 4671-4679; see also U.S. Application No. 20090099115).

a. Antisense Strands

In some embodiments, an oligonucleotide disclosed herein for targeting PCSK9 comprises an antisense strand comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, or 1193-1232. In some embodiments, an oligonucleotide comprises an antisense strand comprising or consisting of at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, or 1193-1232.

In some embodiments, a double-stranded oligonucleotide may have an antisense strand of up to 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have an antisense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, an oligonucleotide may have an antisense strand in a range of 12 to 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, an oligonucleotide may have an antisense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

In some embodiments, an antisense strand of an oligonucleotide may be referred to as a “guide strand.” For example, if an antisense strand can engage with RNA-induced silencing complex (RISC) and bind to an Argonaut protein, or engage with or bind to one or more similar factors, and direct silencing of a target gene, it may be referred to as a guide strand. In some embodiments, a sense strand complementary to a guide strand may be referred to as a “passenger strand.”

b. Sense Strands

In some embodiments, an oligonucleotide disclosed herein for targeting PCSK9 comprises or consists of a sense strand sequence as set forth in in any one of SEQ ID NOs: 1 to 453, 907-1029, and 1153-1192. In some embodiments, an oligonucleotide has a sense strand that comprises or consists of at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in in any one of SEQ ID NOs: 1 to 453, 907-1029, and 1153-1192.

In some embodiments, an oligonucleotide may have a sense strand (or passenger strand) of up to 40 nucleotides in length (e.g., up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand in a range of 12 to 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, an oligonucleotide may have a sense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

In some embodiments, a sense strand comprises a stem-loop structure at its 3′-end. In some embodiments, a sense strand comprises a stem-loop structure at its 5′-end. In some embodiments, a stem is a duplex of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 base pairs in length. In some embodiments, a stem-loop provides the molecule better protection against degradation (e.g., enzymatic degradation) and facilitates targeting characteristics for delivery to a target cell. For example, in some embodiments, a loop provides added nucleotides on which modification can be made without substantially affecting the gene expression inhibition activity of an oligonucleotide. In certain embodiments, an oligonucleotide is provided herein in which the sense strand comprises (e.g., at its 3′-end) a stem-loop set forth as: S₁-L-S₂, in which S₁ is complementary to S₂, and in which L forms a loop between S₁ and S₂ of up to 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). FIG. 3 depicts a non-limiting example of such an oligonucleotide.

In some embodiments, a loop (L) of a stem-loop is a tetraloop (e.g., within a nicked tetraloop structure). A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetraloop has 4 to 5 nucleotides.

c. Duplex Length

In some embodiments, a duplex formed between a sense and antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, a duplex formed between a sense and antisense strand is in the range of 12-30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length). In some embodiments, a duplex formed between a sense and antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments a duplex formed between a sense and antisense strand does not span the entire length of the sense strand and/or antisense strand. In some embodiments, a duplex between a sense and antisense strand spans the entire length of either the sense or antisense strands. In certain embodiments, a duplex between a sense and antisense strand spans the entire length of both the sense strand and the antisense strand.

d. Oligonucleotide Ends

In some embodiments, an oligonucleotide provided herein comprises sense and antisense strands, such that there is a 3′-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand. In some embodiments, oligonucleotides provided herein have one 5′ end that is thermodynamically less stable compared to the other 5′ end. In some embodiments, an asymmetric oligonucleotide is provided that includes a blunt end at the 3′ end of a sense strand and an overhang at the 3′ end of an antisense strand. In some embodiments, a 3′ overhang on an antisense strand is 1-8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length).

Typically, an oligonucleotide for RNAi has a two nucleotide overhang on the 3′ end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, an overhang is a 3′ overhang comprising a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides. However, in some embodiments, the overhang is a 5′ overhang comprising a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides.

In some embodiments, one or more (e.g., 2, 3, 4) terminal nucleotides of the 3′ end or 5′ end of a sense and/or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides of the 3′ end of an antisense strand are modified. In some embodiments, the last nucleotide at the 3′ end of an antisense strand is modified, e.g., comprises 2′-modification, e.g., a 2′-O-methoxyethyl. In some embodiments, the last one or two terminal nucleotides at the 3′ end of an antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3′ end of the antisense strand are not complementary to the target. In some embodiments, the 5′ end and/or the 3′ end of a sense or antisense strand has an inverted cap nucleotide.

e. Mismatches

In some embodiments, there is one or more (e.g., 1, 2, 3, or 4) mismatches between a sense and antisense strand. If there is more than one mismatch between a sense and antisense strand, they may be positioned consecutively (e.g., 2, 3 or more in a row), or interspersed throughout the region of complementarity. In some embodiments, the 3′-terminus of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated at the 3′ terminus of the sense strand. In some embodiments, base mismatches or destabilization of segments at the 3′-end of the sense strand of the oligonucleotide improved the potency of synthetic duplexes in RNAi, possibly through facilitating processing by Dicer.

iii. Single-Stranded Oligonucleotides

In some embodiments, an oligonucleotide for reducing PCSK9 expression as described herein is single-stranded. Such structures may include, but are not limited to single-stranded RNAi oligonucleotides. Recent efforts have demonstrated the activity of single-stranded RNAi oligonucleotides (see, e.g., Matsui et al. (May 2016), Molecular Therapy, Vol. 24(5), 946-955). However, in some embodiments, oligonucleotides provided herein are antisense oligonucleotides (ASOs). An antisense oligonucleotide is a single-stranded oligonucleotide that has a nucleobase sequence which, when written in the 5′ to 3′ direction, comprises the reverse complement of a targeted segment of a particular nucleic acid and is suitably modified (e.g., as a gapmer) so as to induce RNaseH mediated cleavage of its target RNA in cells or (e.g., as a mixmer) so as to inhibit translation of the target mRNA in cells. Antisense oligonucleotides for use in the instant disclosure may be modified in any suitable manner known in the art including, for example, as shown in U.S. Pat. No. 9,567,587, which is incorporated by reference herein for its disclosure regarding modification of antisense oligonucleotides (including, e.g., length, sugar moieties of the nucleobase (pyrimidine, purine), and alterations of the heterocyclic portion of the nucleobase). Further, antisense molecules have been used for decades to reduce expression of specific target genes (see, e.g., Bennett et al.; Pharmacology of Antisense Drugs, Annual Review of Pharmacology and Toxicology, Vol. 57: 81-105).

iv. Oligonucleotide Modifications

Oligonucleotides may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, base-paring properties, RNA distribution and cellular uptake and other features relevant to therapeutic or research use. See, e.g., Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881; Bramsen and Kjems (Frontiers in Genetics, 3 (2012): 1-22). Accordingly, in some embodiments, oligonucleotides of the present disclosure may include one or more suitable modifications. In some embodiments, a modified nucleotide has a modification in its base (or nucleobase), the sugar (e.g., ribose, deoxyribose), or the phosphate group.

The number of modifications on an oligonucleotide and the positions of those nucleotide modifications may influence the properties of an oligonucleotide. For example, oligonucleotides may be delivered in vivo by conjugating them to or encompassing them in a lipid nanoparticle (LNP) or similar carrier. However, when an oligonucleotide is not protected by an LNP or similar carrier (e.g., “naked delivery”), it may be advantageous for at least some of its nucleotides to be modified. Accordingly, in certain embodiments of any of the oligonucleotides provided herein, all or substantially all of the nucleotides of an oligonucleotide are modified. In certain embodiments, more than half of the nucleotides are modified. In certain embodiments, less than half of the nucleotides are modified. Typically, with naked delivery, every nucleotide is modified at the 2′-position of the sugar group of that nucleotide. These modifications may be reversible or irreversible. Typically, the 2′ position modification is a 2′-fluoro, 2′-O-methyl, etc. In some embodiments, an oligonucleotide as disclosed herein has a number and type of modified nucleotides sufficient to cause the desired characteristic (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and/or thermodynamic stability).

a. Sugar Modifications

In some embodiments, a modified sugar (also referred to herein as a sugar analog) includes a modified deoxyribose or ribose moiety, e.g., in which one or more modifications occur at the 2′, 3′, 4′, and/or 5′ carbon position of the sugar. In some embodiments, a modified sugar may also include non-natural alternative carbon structures such as those present in locked nucleic acids (“LNA”) (see, e.g., Koshkin et al. (1998), Tetrahedron 54, 3607-3630), unlocked nucleic acids (“UNA”) (see, e.g., Snead et al. (2013), Molecular Therapy—Nucleic Acids, 2, e103), and bridged nucleic acids (“BNA”) (see, e.g., Imanishi and Obika (2002), The Royal Society of Chemistry, Chem. Commun., 1653-1659). Koshkin et al., Snead et al., and Imanishi and Obika are incorporated by reference herein for their disclosures relating to sugar modifications.

In some embodiments, a nucleotide modification in a sugar comprises a 2′-modification. In some embodiments, the 2′-modification may be 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, or 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. Typically, the modification is 2′-fluoro, 2′-O-methyl, or 2′-O-methoxyethyl. However, a large variety of 2′ position modifications that have been developed for use in oligonucleotides can be employed in oligonucleotides disclosed herein. See, e.g., Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881. In some embodiments, a modification in a sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring. For example, a modification of a sugar of a nucleotide may comprise a linkage between the 2′-carbon and a 1′-carbon or 4′-carbon of the sugar. For example, the linkage may comprise an ethylene or methylene bridge. In some embodiments, a modified nucleotide has an acyclic sugar that lacks a 2′-carbon to 3′-carbon bond. In some embodiments, a modified nucleotide has a thiol group, e.g., in the 4′ position of the sugar.

In some embodiments, the terminal 3′-end group (e.g., a 3′-hydroxyl) is a phosphate group or other group, which can be used, for example, to attach linkers, adapters or labels or for the direct ligation of an oligonucleotide to another nucleic acid.

b. 5′ Terminal Phosphates

5′-terminal phosphate groups of oligonucleotides may or in some circumstances enhance the interaction with Argonaut 2. However, oligonucleotides comprising a 5′-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, oligonucleotides include analogs of 5′ phosphates that are resistant to such degradation. In some embodiments, a phosphate analog may be oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 5′ end of an oligonucleotide strand is attached to a chemical moiety that mimics the electrostatic and steric properties of a natural 5′-phosphate group (“phosphate mimic”) (see, e.g., Prakash et al. (2015), Nucleic Acids Res., Nucleic Acids Res. 2015 Mar. 31; 43(6): 2993-3011, the contents of which relating to phosphate analogs are incorporated herein by reference). Many phosphate mimics have been developed that can be attached to the 5′ end (see, e.g., U.S. Pat. No. 8,927,513, the contents of which relating to phosphate analogs are incorporated herein by reference). Other modifications have been developed for the 5′ end of oligonucleotides (see, e.g., WO 2011/133871, the contents of which relating to phosphate analogs are incorporated herein by reference). In certain embodiments, a hydroxyl group is attached to the 5′ end of the oligonucleotide.

In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”). See, for example, International Patent Application PCT/US2017/049909, filed on Sep. 1, 2017, U.S. Provisional Application Nos. 62/383,207, entitled 4′ Phosphate Analogs and Oligonucleotides Comprising the Same, filed on Sep. 2, 2016, and 62/393,401, filed on Sep. 12, 2016, entitled 4′-Phosphate Analogs and Oligonucleotides Comprising the Same, the contents of each of which relating to phosphate analogs are incorporated herein by reference. In some embodiments, an oligonucleotide provided herein comprises a 4′-phosphate analog at a 5′-terminal nucleotide. In some embodiments, a phosphate analog is an oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. In other embodiments, a 4′-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bound to the 4′-carbon of the sugar moiety or analog thereof. In certain embodiments, a 4′-phosphate analog is an oxymethylphosphonate. In some embodiments, an oxymethylphosphonate is represented by the formula —O—CH₂—PO(OH)₂ or —O—CH₂—PO(OR)₂, in which R is independently selected from H, CH₃, an alkyl group, CH₂CH₂CN, CH₂OCOC(CH₃)₃, CH₂OCH₂CH₂Si(CH₃)₃, or a protecting group. In certain embodiments, the alkyl group is CH₂CH₃. More typically, R is independently selected from H, CH₃, or CH₂CH₃.

c. Modified Internucleoside Linkages

In some embodiments, the oligonucleotide may comprise a modified internucleoside linkage. In some embodiments, phosphate modifications or substitutions may result in an oligonucleotide that comprises at least one (e.g., at least 1, at least 2, at least 3, at least 4, or at least 5) modified internucleotide linkage. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1 to 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

A modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage. In some embodiments, at least one modified internucleotide linkage of any one of the oligonucleotides as disclosed herein is a phosphorothioate linkage

d. Base Modifications

In some embodiments, oligonucleotides provided herein have one or more modified nucleobases. In some embodiments, modified nucleobases (also referred to herein as base analogs) are linked at the 1′ position of a nucleotide sugar moiety. In certain embodiments, a modified nucleobase is a nitrogenous base. In certain embodiments, a modified nucleobase does not contain a nitrogen atom. See e.g., U.S. Published Patent Application No. 20080274462. In some embodiments, a modified nucleotide comprises a universal base. However, in certain embodiments, a modified nucleotide does not contain a nucleobase (abasic).

In some embodiments, a universal base is a heterocyclic moiety located at the 1′ position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering the structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., oligonucleotide) that is fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower T_(m) than a duplex formed with the complementary nucleic acid. However, in some embodiments, compared to a reference single-stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher T_(m) than a duplex formed with the nucleic acid comprising the mismatched base.

Non-limiting examples of universal-binding nucleotides include inosine, ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct. 11; 22(20):4039-43. Each of the foregoing is incorporated by reference herein for their disclosures relating to base modifications).

e. Reversible Modifications

While certain modifications to protect an oligonucleotide from the in vivo environment before reaching target cells can be made, they can reduce the potency or activity of the oligonucleotide once it reaches the cytosol of the target cell. Reversible modifications can be made such that the molecule retains desirable properties outside of the cell, which are then removed upon entering the cytosolic environment of the cell. Reversible modification can be removed, for example, by the action of an intracellular enzyme or by the chemical conditions inside of a cell (e.g., through reduction by intracellular glutathione).

In some embodiments, a reversibly modified nucleotide comprises a glutathione-sensitive moiety. Typically, nucleic acid molecules have been chemically modified with cyclic disulfide moieties to mask the negative charge created by the internucleotide diphosphate linkages and improve cellular uptake and nuclease resistance. See U.S. Published Application No. 2011/0294869 originally assigned to Traversa Therapeutics, Inc. (“Traversa”), PCT Publication No. WO 2015/188197 to Solstice Biologics, Ltd. (“Solstice”), Meade et al., Nature Biotechnology, 2014, 32:1256-1263 (“Meade”), PCT Publication No. WO 2014/088920 to Merck Sharp & Dohme Corp, each of which are incorporated by reference for their disclosures of such modifications. This reversible modification of the internucleotide diphosphate linkages is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g. glutathione). Earlier examples include neutralizing phosphotriester modifications that were reported to be cleavable inside cells (Dellinger et al. J. Am. Chem. Soc. 2003, 125:940-950).

In some embodiments, such a reversible modification allows protection during in vivo administration (e.g., transit through the blood and/or lysosomal/endosomal compartments of a cell) where the oligonucleotide will be exposed to nucleases and other harsh environmental conditions (e.g., pH). When released into the cytosol of a cell where the levels of glutathione are higher compared to extracellular space, the modification is reversed and the result is a cleaved oligonucleotide. Using reversible, glutathione sensitive moieties, it is possible to introduce sterically larger chemical groups into the oligonucleotide of interest as compared to the options available using irreversible chemical modifications. This is because these larger chemical groups will be removed in the cytosol and, therefore, should not interfere with the biological activity of the oligonucleotides inside the cytosol of a cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to modify the kinetics of its release.

In some embodiments, a glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, a glutathione-sensitive moiety is attached to the 2′-carbon of the sugar of a modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5′-carbon of a sugar, particularly when the modified nucleotide is the 5′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3′-carbon of a sugar, particularly when the modified nucleotide is the 3′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, e.g., International Patent Application PCT/US2017/048239, which published on Mar. 1, 2018 as International Patent Publication WO2018/039364, entitled Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof, which was filed on Aug. 23, 2016, the contents of which are incorporated by reference herein for its relevant disclosures.

v. Targeting Ligands

In some embodiments, it may be desirable to target the oligonucleotides of the disclosure to one or more cells or one or more organs. Such a strategy may help to avoid undesirable effects in other organs, or may avoid undue loss of the oligonucleotide to cells, tissue or organs that would not benefit for the oligonucleotide. Accordingly, in some embodiments, oligonucleotides disclosed herein may be modified to facilitate targeting of a particular tissue, cell or organ, e.g., to facilitate delivery of the oligonucleotide to the liver. In certain embodiments, oligonucleotides disclosed herein may be modified to facilitate delivery of the oligonucleotide to the hepatocytes of the liver. In some embodiments, an oligonucleotide comprises a nucleotide that is conjugated to one or more targeting ligands.

A targeting ligand may comprise a carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein or part of a protein (e.g., an antibody or antibody fragment) or lipid. In some embodiments, a targeting ligand is an aptamer. For example, a targeting ligand may be an RGD peptide that is used to target tumor vasculature or glioma cells, CREKA peptide to target tumor vasculature or stoma, transferrin, lactoferrin, or an aptamer to target transferrin receptors expressed on CNS vasculature, or an anti-EGFR antibody to target EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5′ or 3′ end of the sense or antisense strand) such that the targeting ligands resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5′ or 3′ end of the sense strand and 1, 2, 3, or 4 nucleotides of the loop of the stem may be individually conjugated to a targeting ligand, as described, for example, in International Patent Application Publication WO 2016/100401, which was published on Jun. 23, 2016, the relevant contents of which are incorporated herein by reference.

In some embodiments, it is desirable to target an oligonucleotide that reduces the expression of PCSK9 to the hepatocytes of the liver of a subject. Any suitable hepatocyte targeting moiety may be used for this purpose.

GalNAc is a high affinity ligand for asialoglycoprotein receptor (ASGPR), which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to oligonucleotides of the instant disclosure may be used to target these oligonucleotides to the ASGPR expressed on these hepatocyte cells.

In some embodiments, an oligonucleotide of the instant disclosure is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., is conjugated to 2, 3, or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, an oligonucleotide of the instant disclosure is conjugated to one or more bivalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties.

In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of the loop (L) of the stem-loop are each conjugated to a separate GalNAc. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5′ or 3′ end of the sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5′ or 3′ end of the sense strand and 1, 2, 3, or 4 nucleotides of the loop of the stem may be individually conjugated to a GalNAc moiety. In some embodiments, GalNAc moieties are conjugated to a nucleotide of the sense strand. For example, four GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand, where each GalNAc moiety is conjugated to one nucleotide.

Appropriate methods or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication Number WO2016100401 A1, which published on Jun. 23, 2016, and the contents of which relating to such linkers are incorporated herein by reference. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is fairly stable. In some embodiments, a duplex extension (up to 3, 4, 5, or 6 base pairs in length) is provided between a targeting ligand (e.g., a GalNAc moiety) and a double-stranded oligonucleotide.

III. Formulations

Various formulations have been developed to facilitate oligonucleotide use. For example, oligonucleotides can be delivered to a subject or a cellular environment using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the oligonucleotides in the formulation. In some embodiments, provided herein are compositions comprising oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) to reduce the expression of PCSK9. Such compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient portion of the oligonucleotides enters the cell to reduce PCSK9 expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for the reduction of PCSK9 as disclosed herein. In some embodiments, an oligonucleotide is formulated in buffer solutions such as phosphate-buffered saline solutions, liposomes, micellar structures, and capsids. In some embodiments, naked oligonucleotides or conjugates thereof are formulated in water or in an aqueous solution (e.g., water with pH adjustments). In some embodiments, naked oligonucleotides or conjugates thereof are formulated in basic buffered aqueous solutions (e.g., PBS)

Formulations of oligonucleotides with cationic lipids can be used to facilitate transfection of the oligonucleotides into cells. For example, cationic lipids, such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used according to the manufacturer's instructions.

Accordingly, in some embodiments, a formulation comprises a lipid nanoparticle. In some embodiments, an excipient comprises a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2013).

In some embodiments, formulations as disclosed herein comprise an excipient. In some embodiments, an excipient confers to a composition improved stability, improved absorption, improved solubility and/or therapeutic enhancement of the active ingredient. In some embodiments, an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, an oligonucleotide is lyophilized for extending its shelf-life and then made into a solution before use (e.g., administration to a subject). Accordingly, an excipient in a composition comprising any one of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone), or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotides in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

In some embodiments, a composition may contain at least about 0.1% of the therapeutic agent (e.g., an oligonucleotide for reducing PCSK9 expression) or more, although the percentage of the active ingredient(s) may be between about 1% and about 80% or more of the weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

Even though a number of embodiments are directed to liver-targeted delivery of any of the oligonucleotides disclosed herein, targeting of other tissues is also contemplated.

IV. Methods of Use

i. Reducing PCSK9 Expression in Cells

In some embodiments, methods are provided for delivering to a cell an effective amount any one of oligonucleotides disclosed herein for purposes of reducing expression of PCSK9 in the cell. Methods provided herein are useful in any appropriate cell type. In some embodiments, a cell is any cell that expresses PCSK9 (e.g., liver, lung, kidney, spleen, testis, adipose, and intestinal cells). In some embodiments, the cell is a primary cell that has been obtained from a subject and that may have undergone a limited number of a passages, such that the cell substantially maintains its natural phenotypic properties. In some embodiments, a cell to which the oligonucleotide is delivered is ex vivo or in vitro (i.e., can be delivered to a cell in culture or to an organism in which the cell resides). In specific embodiments, methods are provided for delivering to a cell an effective amount any one of the oligonucleotides disclosed herein for purposes of reducing expression of PCSK9 solely or primarily in hepatocytes.

In some embodiments, oligonucleotides disclosed herein can be introduced using appropriate nucleic acid delivery methods including injection of a solution containing the oligonucleotides, bombardment by particles covered by the oligonucleotides, exposing the cell or organism to a solution containing the oligonucleotides, or electroporation of cell membranes in the presence of the oligonucleotides. Other appropriate methods for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and others.

The consequences of inhibition can be confirmed by an appropriate assay to evaluate one or more properties of a cell or subject, or by biochemical techniques that evaluate molecules indicative of PCSK9 expression (e.g., RNA, protein). In some embodiments, the extent to which an oligonucleotide provided herein reduces levels of expression of PCSK9 is evaluated by comparing expression levels (e.g., mRNA or protein levels of PCSK9 to an appropriate control (e.g., a level of PCSK9 expression in a cell or population of cells to which an oligonucleotide has not been delivered or to which a negative control has been delivered). In some embodiments, an appropriate control level of PCSK9 expression may be a predetermined level or value, such that a control level need not be measured every time. The predetermined level or value can take a variety of forms. In some embodiments, a predetermined level or value can be single cut-off value, such as a median or mean.

In some embodiments, administration of an oligonucleotide as described herein results in a reduction in the level of PCSK9 expression in a cell. In some embodiments, the reduction in levels of PCSK9 expression may be a reduction to 1% or lower, 5% or lower, 10% or lower, 15% or lower, 20% or lower, 25% or lower, 30% or lower, 35% or lower, 40% or lower, 45% or lower, 50% or lower, 55% or lower, 60% or lower, 70% or lower, 80% or lower, or 90% or lower compared with an appropriate control level of PCSK9. The appropriate control level may be a level of PCSK9 expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein. In some embodiments, the effect of delivery of an oligonucleotide to a cell according to a method disclosed herein is assessed after a finite period of time. For example, levels of PCSK9 may be analyzed in a cell at least 8 hours, 12 hours, 18 hours, 24 hours; or at least one, two, three, four, five, six, seven, or fourteen days after introduction of the oligonucleotide into the cell.

In some embodiments, an oligonucleotide is delivered in the form of a transgene that is engineered to express in a cell the oligonucleotides disclosed herein (e.g., in the form of an shRNA). In some embodiments, an oligonucleotide is delivered using a transgene that is engineered to express any oligonucleotide disclosed herein. Transgenes may be delivered using viral vectors (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus or herpes simplex virus) or non-viral vectors (e.g., plasmids or synthetic mRNAs). In some embodiments, transgenes can be injected directly to a subject.

ii. Treatment Methods

Aspects of the disclosure relate to methods for reducing PCSK9 expression for the treatment of hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof in a subject. In some embodiments, the methods may comprise administering to a subject in need thereof an effective amount of any one of the oligonucleotides disclosed herein. In some embodiments, such treatments may be used, for example, to decrease or prevent hypercholesterolemia (high levels of low density lipoprotein (LDL)-cholesterol), atherosclerosis, coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke (i.e., death of brain cells resulting from insufficient blood and oxygen flow to the brain), feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, loss of consciousness, peripheral artery disease, and/or kidney problems (e.g., chronic kidney disease). In some embodiments, such treatments may be used, for example, to treat or prevent one or more symptoms associated with hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof.

Accordingly, in some embodiments, the present disclosure provides methods of treating a subject at risk of (or susceptible to) hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof including coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke (i.e., death of brain cells resulting from insufficient blood and oxygen flow to the brain), feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, loss of consciousness, peripheral artery disease, and/or kidney problems (e.g., chronic kidney disease).

In certain aspects, the disclosure provides a method for preventing in a subject, a disease, disorder, symptom, or condition as described herein by administering to the subject a therapeutic agent (e.g., an oligonucleotide or vector or transgene encoding same). In some embodiments, the subject to be treated is a subject who will benefit therapeutically from a reduction in the amount of PCSK9 protein, e.g., in the liver.

Methods described herein typically involve administering to a subject an effective amount of an oligonucleotide, that is, an amount capable of producing a desirable therapeutic result. A therapeutically acceptable amount may be an amount that is capable of treating a disease or disorder. The appropriate dosage for any one subject will depend on certain factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.

In some embodiments, a subject is administered any one of the compositions disclosed herein either enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intramuscular injection), topically (e.g., epicutaneous, inhalational, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the liver of a subject). Typically, oligonucleotides disclosed herein are administered intravenously or subcutaneously.

In some embodiments, oligonucleotides are administered at a dose in a range of 0.1 mg/kg to 25 mg/kg (e.g., 1 mg/kg to 5 mg/kg). In some embodiments, oligonucleotides are administered at a dose in a range of 0.1 mg/kg to 5 mg/kg or in a range of 0.5 mg/kg to 5 mg/kg.

As a non-limiting set of examples, the oligonucleotides of the instant disclosure would typically be administered once per year, twice per year, quarterly (once every three months), bi-monthly (once every two months), monthly, or weekly.

In some embodiments, the subject to be treated is a human (e.g., a human patient) or non-human primate or other mammalian subject. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

EXAMPLES Example 1: Development of PCSK9 Oligonucleotide Inhibitors Using Human and Mouse Cell-Based Assays

Human and mouse-based assays were used to develop candidate oligonucleotides for inhibition of PCSK9 expression. First, a computer-based algorithm was used to generate candidate oligonucleotide sequences (25-27-mer) for PCSK9 inhibition. Cell-based assays and PCR assays were then employed for evaluation of candidate oligonucleotides for their ability to reduce PCSK9 expression.

The computer-based algorithm provided oligonucleotides that were complementary to human PCSK9 mRNA (SEQ ID NO: 1245, Table 1), of which certain sequences were also complementary to Rhesus monkey PCSK9 mRNA (SEQ ID NO: 1246, Table 1).

TABLE 1 Sequences of human and Rhesus monkey PCSK9 mRNA Species GenBank RefSeq # SEQ ID NO. Human NM_174936.3 1245 Rhesus monkey NM_001112660.1 1246

Of the oligonucleotides that the algorithm provided, 576 oligonucleotides were selected as candidates for experimental evaluation in a Huh-7 cell-based assay. In this assay, Huh-7 human liver cells stably expressing PCSK9 were transfected with the oligonucleotides. Cells were maintained for a period of time following transfection and then levels of remaining PCSK9 mRNA were interrogated using TAQMAN®-based qPCR assays. Two qPCR assays, a 3′ assay and a 5′ assay, were used to determine mRNA levels as measured by HEX (housekeeping gene—SFRS9) and FAM probes, respectively. The results of the cell-based assay with the 576 oligonucleotides are shown in FIGS. 1A and 1B. The percent mRNA remaining is shown for each of the 5′ assay (circle shapes) and the 3′ assay (diamond shapes) in FIG. 1B. Oligonucleotides with the lowest percentage of mRNA remaining compared to mock transfection controls were considered hits. Oligonucleotides with low complementarity to the human genome were used as negative controls.

Based on the activity and locations of these oligonucleotides, hotspots on the human PCSK9 mRNA were defined. A hotspot was identified as a stretch on the human PCSK9 mRNA sequence associated with at least one oligonucleotide resulting in mRNA levels that were less than or equal to 35% in either assay compared with controls. Accordingly, the following hotspots within the human PCSK9 mRNA sequence (NM_174936.3) were identified: 746-783, 2602-2639, 2737-2792, 2880-2923, 2956-2996, 3015-3075, 3099-3178, 3190-3244, 3297-3359, 3649-3446, 3457-3499, and 3532-3715.

The sequences of the hotspots are outlined in Table 2.

TABLE 2 Sequences of Hotspots Hotspot Position In Human PCSK9 SEQ mRNA Sequence ID NO. 746-783 CGACCTGCTGGAGCTGGCCTTGAAGTTGCC 1233 CCATGTCG 2602-2639 AGCCTCCTTGCCTGGAACTCACTCACTCTG 1234 GGTGCCTC 2737-2792 CAATGTGCCGATGTCCGTGGGCAGAATGAC 1235 TTTTATTGAGCTCTTGTTCCGTGCCA 2880-2923 CGTTGGGGGGTGAGTGTGAAAGGTGCTGAT 1236 GGCCCTCATCTCCA 2956-2996 GATTAATGGAGGCTTAGCTTTCTGGATGGC 1237 ATCTAGCCAGA 3015-3075 CCCTGGTGGTCACAGGCTGTGCCTTGGTTT 1238 CCTGAGCCACCTTTACTCTGCTCTATGCCA G 3099-3178 TGGCCTGCGGGGAGCCATCACCTAGGACTG 1239 ACTCGGCAGTGTGCAGTGGTGCATGCACTG TCTCAGCCAACCCGCTCCAC 3190-3244 GTACACATTCGCACCCCTACTTCACAGAGG 1240 AAGAAACCTGGAACCAGAGGGGGCG 3297-3359 GCTCTGAAGCCAAGCCTCTTCTTACTTCAC 1241 CCGGCTGGGCTCCTCATTTTTACGGGTAAC AGT 3469-3446 AACGATGCCTGCAGGCATGGAACTTTTTCC 1242 GTTATCACCCAGGCCT 3457-3499 TTCACTGGCCTGGCGGAGATGCTTCTAAGG 1243 CATGGTCGGGGGA 3532-3715 GCCCCACCCAAGCAAGCAGACATTTATCTT 1244 TTGGGTCTGTCCTCTCTGTTGCCTTTTTAC AGCCAACTTTTCTAGACCTGTTTTGCTTTT GTAACTTGAAGATATTTATTCTGGGTTTTG TAGCATTTTTATTAATATGGTGACTTTTTA AAATAAAAACAAACAAACGTTGTCCTAACA AAAA

Dose Response Analysis

Of the 576 oligonucleotides evaluated in the initial Huh-7 cell-based assay, 96 particularly active oligonucleotides were selected as hits based on their ability to knock down PCSK9 levels and were subjected to a secondary screen (FIGS. 2A and 2B).

In this secondary screen, the candidate oligonucleotides were tested using the same assay as in the primary screen, but at two different concentrations 0.1 nM and 1 nM (FIGS. 2A and 2B). The target mRNA levels were generally normalized based on splicing factor, arginine/serine-rich 9 (SFRS9), a housekeeping gene that provides a stable expression reference across samples, to generate the percent mRNA shown in FIGS. 2A and 2B. The tested oligonucleotides in each of FIGS. 2A and 2B are shown compared to mock transfection control. All 96 oligonucleotides had the same modification pattern, designated M1, which contains a combination of ribonucleotides, deoxyribonucleotides and 2′-O-methyl modified nucleotides. The sequences of the 96 oligonucleotides tested are provided in Table 3.

TABLE 3 Candidate oligonucleotide Sequences for Huh-7 Cell-Based Assay Sense Corresponding Antisense SEQ ID NO. SEQ ID NO. 35, 41, 51, 53, 56-58, 66, 177- 488, 494, 504, 506, 509-511, 180, 187, 192, 196, 201-204, 519, 630-633, 640, 645, 649, 219-225, 227, 237-241, 243, 654-657, 672-678, 680, 690- 248, 249, 257, 261, 262, 264, 694, 696, 701, 702, 710, 714, 266, 268, 274, 280, 281, 288- 715, 717, 719, 721, 727, 733, 292, 297, 304-306, 315, 316, 734, 741-745, 750, 757-759, 320-322, 328-330, 333, 334, 768, 769, 773-775, 781-783, 344, 345, 347, 349, 351, 352, 786, 787, 797, 798, 800, 802, 374, 375, 385-395, 400-402, 804, 805, 827, 828, 838-848, 405, 408-411, 418, 433, 434, 853-855, 858, 861-864, 871, 440-442 886, 887, 893-895 Sense and antisense SEQ ID NO. columns provide the sense strand and respective antisense strand, in relative order, that are hybridized to make each oligonucleotide. For example, sense strand of SEQ ID NO: 35 hybridizes with antisense strand of SEQ ID NO: 488; each of the oligonucleotides tested had the same modification pattern.

At this stage, the most potent sequences from the testing were selected for further analysis. The selected sequences were converted to a nicked tetraloop conjugate structure format (a 36-mer passenger strand with a 22-mer guide strand). See FIG. 3 for a generic tetraloop conjugate structure. Four GalNAc moieties were conjugated to nucleotides in the tetraloop of the sense strand. Conjugation was performed using a click linker. The GalNAc used was as shown below:

These oligonucleotides were then tested as before, and each oligonucleotide was evaluated at two concentrations for its ability to reduce PCSK9 mRNA expression in vitro, using Huh-7 cells, as well as in vivo, using a mouse HDI model.

In Vivo Murine Screening and In Vitro Human Cell Line Screening

Data from the above in vitro experiments were assessed to identify tetraloops and modification patterns that would improve delivery properties while maintaining activity for reduction of PCSK9 expression in the mouse hepatocytes. As shown in FIG. 4 , 12 human PCSK9 tetraloop conjugates with a range of modifications were dosed subcutaneously into mice at a concentration of 3 mg/kg. Animals were administered 2 ml of human PCSK9 plasmid (pcDNA3.1-hPCSK9, total 16 μg) suspended in PBS per animal by tail vein (intravenous) injection on day 3 after the subcutaneous dosing of tetraloop conjugates. Mice were euthanized on day 4 following administration. Liver samples were obtained and RNA was extracted to evaluate PCSK9 mRNA levels by RT-qPCR. The percent PCSK9 mRNA as compared to PBS control mRNA was determined based on these measurements.

Further tetraloop sequences were tested in human Huh-7 cells at two different concentrations (0.03 nM and 0.1 nM in tetraloop formation; labeled as “Phase T2”) (FIG. 5A). From the 40 tetraloop oligonucleotides tested (shown in FIG. 5A), 21 different base sequences were selected to be scaled up as 5′-MOP/GalNAc conjugates for further in vivo testing (FIGS. 5B and 5C). The PCSK9 oligonucleotides were subcutaneously administered to CD-1 mice transiently expressing human PCSK9 mRNA by hydrodynamic injection (HDI) of a human PCSK9 expression plasmid (pcDNA3.1-hPCSK9, total 16 μg). Mice were euthanized on day 4 following administration. Liver samples were obtained and RNA was extracted to evaluate PCSK9 mRNA levels by RT-qPCR. The percent PCSK9 mRNA as compared to PBS control mRNA was determined based on these measurements. As shown in FIGS. 5B-5C, different concentrations (1 mg/kg and 2 mg/kg) were used for the candidate molecules. A candidate of sense sequence SEQ ID NO: 1182 and antisense sequence SEQ ID NO: 1222 may be seen in both FIG. 5B and FIG. 5C.

Additional testing of certain PCSK9 oligonucleotides in the mouse HDI model described above was performed using three different PCSK9 tetraloop conjugates with varied modification patterns at three different concentrations (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg). Results are shown in FIGS. 6A and 6B.

In Vivo Non-Human Primate Screening

An additional study was performed to evaluate PCSK9 mRNA KD with tetraloop conjugates in non-human primates. Cynomolgus monkeys (n=4 per group) were administered 3 or 6 mg/kg subcutaneously in a single dose. Clinical observations were recorded daily, and blood samples were taken three times prior to the dosing and twice a week until day 36 and weekly through day 90. Serum samples were analyzed for a standard LFT panel (ALT, AST, ALP, and GGT), as well as LDL-c, HDL-c, total cholesterol, and TG. Three sets of sequences (sense and antisense) were tested: S1266-AS1269, S1267-AS1270, and S1268-AS1271 and results are shown in FIGS. 7A-7C. All three sets of sequences were able to reduce plasma levels of PCSK9 relative to the pre-dose levels.

Materials and Methods Transfection

For the first screen, Lipofectamine RNAiMAX™ was used to complex the oligonucleotides for efficient transfection. Oligonucleotides, RNAiMAX and Opti-MEM incubated together at room temperature for 20 minutes and then 50 μL of this mix was added per well to plates prior to transfection. Media was aspirated from a flask of actively passaging cells and the cells were incubated at 37° C. in the presence of trypsin for 3-5 minutes. After cells no longer adhered to the flask, cell growth media (lacking penicillin and streptomycin) was added to neutralize the trypsin and to suspend the cells. A 10 μL aliquot was removed and cells were counted with a hemocytometer to quantify the cells on a per milliliter basis. A diluted cell suspension was added to the 96-well transfection plates, which already contained the oligonucleotides in Opti-MEM. The transfection plates were then incubated for 24 hours at 37° C. After 24 hours of incubation, media was aspirated from each well.

For subsequent screens and experiments, e.g., the secondary screen, Lipofectamine RNAiMAX was used to complex the oligonucleotides for reverse transfection. The complexes were made by mixing RNAiMAX and siRNAs in OptiMEM medium for 15 minutes. The transfection mixture was transferred to multi-well plates and cell suspension was added to the wells. After 24 hours incubation the cells were washed once with PBS and then processed described above.

Hydrodynamic Injection (HDI)

CD-1 female mice were obtained from Charles River Laboratories. All mice were maintained in an AALAC and IACUC approved animal facility at the Dicerna Pharmaceuticals. Animals were divided into appropriate number of study groups and dosed with the test article assigned to that group. Animals were dosed subcutaneously with the PCSK tetraloop conjugates. Animals were administered with 2 ml hPCSK9 plasmid suspended in PBS per animal by tail vein intravenous injection on day 3 after the subcutaneous dosing of tetraloop conjugate. Mice were sacrificed on days 4 via CO₂ asphyxiation and liver tissue was collected. Liver tissue was collected by taking two 4 mm punch biopsies and processed to RNA isolation, cDNA synthesis, q-RT PCR, according the manufacturer's protocol. pcDNA3.1-hPCSK9 plasmid encoding the human PCSK9 (NM 174936.3) gene (hPCSK9) was synthesized by Genewiz.

cDNA Synthesis

Cells were lysed for 5 minutes using the iScript RT-qPCR sample preparation buffer from Bio-Rad. The supernatants containing total RNA were then stored at −80° C. or used for reverse transcription using the High Capacity Reverse Transcription kit (Life Technologies) in a 10 microliter reaction. The cDNA was then diluted to 50 μL with nuclease free water and used for quantitative PCR with multiplexed 5′-endonuclease assays and SSoFast qPCR mastermix (Bio-Rad laboratories).

qPCR Assays

For each target, mRNA levels were quantified by two 5′ nuclease assays. In general, several assays are screened for each target. The two assays selected displayed a combination of good efficiency, low limit of detection, and broad 5′43′ coverage of the gene of interest (GOI). Both assays against one GOI could be combined in one reaction when different fluorophores were used on the respective probes. Thus, the final step in assay validation was to determine the efficiency of the selected assays when they were combined in the same qPCR or “multi-plexed.”

Linearized plasmids for both assays in 10-fold dilutions were combined and qPCR was performed. The efficiency of each assay was determined as described above. The accepted efficiency rate was 90-110%.

While validating multi-plexed reactions using linearized plasmid standards, Cq values for the target of interest were also assessed using cDNA as the template. The cDNA, in this case, was derived from RNA isolated on the Corbett (˜5 ng/μl in water) from untransfected cells. In this way, the observed Cq values from this sample cDNA were representative of the expected Cq values from a 96-well plate transfection. In cases where Cq values were greater than 30, other cell lines were sought that exhibit higher expression levels of the gene of interest. A library of total RNA isolated from via high-throughput methods on the Corbett from each human and mouse line was generated and used to screen for acceptable levels of target expression.

Description of Oligonucleotide Nomenclature

All oligonucleotides described herein are designated either SN₁-ASN₂-MN₃. The following designations apply:

-   -   N₁: sequence identifier number of the sense strand sequence     -   N₂: sequence identifier number of the antisense strand sequence     -   N₃: reference number of modification pattern, in which each         number represents a pattern of modified nucleotides in the         oligonucleotide.         For example, S1-AS454-M1 represents an oligonucleotide with a         sense sequence that is set forth by SEQ ID NO: 1, an antisense         sequence that is set forth by SEQ ID NO: 454, and which is         adapted to a modification pattern identified as M1.

TABLE 4 S SEQ AS SEQ App Name Sense Sequence/mRNA seq ID NO Antisense Sequence ID NO S1-AS454- AAGCACCCACACCCUAGAAUGUUTC    1 GAAACAUUCUAGGGUGUGG  454 M1 GUGCUUGA S2-AS455- AGCACCCACACCCUAGAAGUUUUCC    2 GGAAAACUUCUAGGGUGUG  455 M1 GGUGCUUG S3-AS456- GCACCCACACCCUAGAAGGUUUCCG    3 CGGAAACCUUCUAGGGUGU  456 M1 GGGUGCUU S4-AS457- ACCCACACCCUAGAAGGUUUCCGCA    4 UGCGGAAACCUUCUAGGGU  457 M1 GUGGGUGC S5-AS458- CCCACACCCUAGAAGGUUUUCGCAG    5 CUGCGAAAACCUUCUAGGG  458 M1 UGUGGGUG S6-AS459- AGUUCAGGGUCUGAGCCUGUAGGAG    6 CUCCUACAGGCUCAGACCC  459 M1 UGAACUGA S7-AS460- GUUCAGGGUCUGAGCCUGGAGGAGT    7 ACUCCUCCAGGCUCAGACC  460 M1 CUGAACUG S8-AS461- UUCAGGGUCUGAGCCUGGAUGAGTG    8 CACUCAUCCAGGCUCAGAC  461 M1 CCUGAACU S9-AS462- UCAGGGUCUGAGCCUGGAGUAGUGA    9 UCACUACUCCAGGCUCAGA  462 M1 CCCUGAAC S10-AS463- AGGGUCUGAGCCUGGAGGAUUGAGC   10 GCUCAAUCCUCCAGGCUCA  463 M1 GACCCUGA S11-AS464- GGUCUGAGCCUGGAGGAGUUAGCCA   11 UGGCUAACUCCUCCAGGCU  464 M1 CAGACCCU S12-AS465- AGGAUUCCGCGCGCCCCUUUACGCG   12 CGCGUAAAGGGGCGCGCGG  465 M1 AAUCCUGG S13-AS466- GGAUUCCGCGCGCCCCUUCACGCGC   13 GCGCGUGAAGGGGCGCGCG  466 M1 GAAUCCUG S14-AS467- UCACGCGCCCUGCUCCUGAACUUCA   14 UGAAGUUCAGGAGCAGGGC  467 M1 GCGUGAAG S15-AS468- CACGCGCCCUGCUCCUGAAUUUCAG   15 CUGAAAUUCAGGAGCAGGG  468 M1 CGCGUGAA S16-AS469- CCCUGCUCCUGAACUUCAGUUCCTG   16 CAGGAACUGAAGUUCAGGA  469 M1 GCAGGGCG S17-AS470- CUGCUCCUGAACUUCAGCUUCUGCA   17 UGCAGAAGCUGAAGUUCAG  470 M1 GAGCAGGG S18-AS471- UGCUCCUGAACUUCAGCUCUUGCAC   18 GUGCAAGAGCUGAAGUUCA  471 M1 GGAGCAGG S19-AS472- GCUCCUGAACUUCAGCUCCUGCACA   19 UGUGCAGGAGCUGAAGUUC  472 M1 AGGAGCAG S20-AS473- CUCCUGAACUUCAGCUCCUUCACAG   20 CUGUGAAGGAGCUGAAGUU  473 M1 CAGGAGCA S21-AS474- UCCUGAACUUCAGCUCCUGUACAGT   21 ACUGUACAGGAGCUGAAGU  474 M1 UCAGGAGC S22-AS475- CCUGAACUUCAGCUCCUGCACAGTC   22 GACUGUGCAGGAGCUGAAG  475 M1 UUCAGGAG S23-AS476- CUGAACUUCAGCUCCUGCAUAGUCC   23 GGACUAUGCAGGAGCUGAA  476 M1 GUUCAGGA S24-AS477- UGAACUUCAGCUCCUGCACAGUCCT   24 AGGACUGUGCAGGAGCUGA  477 M1 AGUUCAGG S25-AS478- GAACUUCAGCUCCUGCACAUUCCTC   25 GAGGAAUGUGCAGGAGCUG  478 M1 AAGUUCAG S26-AS479- AACUUCAGCUCCUGCACAGUCCUCC   26 GGAGGACUGUGCAGGAGCU  479 M1 GAAGUUCA S27-AS480- ACUUCAGCUCCUGCACAGUUCUCCC   27 GGGAGAACUGUGCAGGAGC  480 M1 UGAAGUUC S28-AS481- CUUCAGCUCCUGCACAGUCUUCCCC   28 GGGGAAGACUGUGCAGGAG  481 M1 CUGAAGUU S29-AS482- ACAGUCCUCCCCACCGCAAUGCUCA   29 UGAGCAUUGCGGUGGGGAG  482 M1 GACUGUGC S30-AS483- CAGUCCUCCCCACCGCAAGUCUCAA   30 UUGAGACUUGCGGUGGGGA  483 M1 GGACUGUG S31-AS484- GCCUCUAGGUCUCCUCGCCAGGACA   31 UGUCCUGGCGAGGAGACCU  484 M1 AGAGGCCG S32-AS485- GCCAGGACAGCAACCUCUCUCCUGG   32 CCAGGAGAGAGGUUGCUGU  485 M1 CCUGGCGA S33-AS486- GGACAGCAACCUCUCCCCUUGCCCT   33 AGGGCAAGGGGAGAGGUUG  486 M1 CUGUCCUG S34-AS487- CCCCUGGCCCUCAUGGGCAUCGUCA   34 UGACGAUGCCCAUGAGGGC  487 M1 CAGGGGAG S35-AS488- UGGCCCUCAUGGGCACCGUUAGCTC   35 GAGCUAACGGUGCCCAUGA  488 M1 GGGCCAGG S36-AS489- GGCCCUCAUGGGCACCGUCAGCUCC   36 GGAGCUGACGGUGCCCAUG  489 M1 AGGGCCAG S37-AS490- GCCCUCAUGGGCACCGUCAUCUCCA   37 UGGAGAUGACGGUGCCCAU  490 M1 GAGGGCCA S38-AS491- GCGGUCCUGGUGGCCGCUGUCACTG   38 CAGUGACAGCGGCCACCAG  491 M1 GACCGCCU S39-AS492- GGCCUGGCCGAAGCACCCGAGCACG   39 CGUGCUCGGGUGCUUCGGC  492 M1 CAGGCCGU S40-AS493- ACCCGAGCACGGAACCACAUCCACC   40 GGUGGAUGUGGUUCCGUGC  493 M1 UCGGGUGC S41-AS494- AGCACGGAACCACAGCCACUUUCCA   41 UGGAAAGUGGCUGUGGUUC  494 M1 CGUGCUCG S42-AS495- CACGGAACCACAGCCACCUUCCACC   42 GGUGGAAGGUGGCUGUGGU  495 M1 UCCGUGCU S43-AS496- ACGGAACCACAGCCACCUUUCACCG   43 CGGUGAAAGGUGGCUGUGG  496 M1 UUCCGUGC S44-AS497- GCCAAGGAUCCGUGGAGGUUGCCTG   44 CAGGCAACCUCCACGGAUC  497 M1 CUUGGCGC S45-AS498- CCAAGGAUCCGUGGAGGUUUCCUGG   45 CCAGGAAACCUCCACGGAU  498 M1 CCUUGGCG S46-AS499- AAGGAUCCGUGGAGGUUGCUUGGCA   46 UGCCAAGCAACCUCCACGG  499 M1 AUCCUUGG S47-AS500- GGAUCCGUGGAGGUUGCCUUGCACC   47 GGUGCAAGGCAACCUCCAC  500 M1 GGAUCCUU S48-AS501- UGGAGGUUGCCUGGCACCUACGUGG   48 CCACGUAGGUGCCAGGCAA  501 M1 CCUCCACG S49-AS502- UGCCUGGCACCUACGUGGUUGUGCT   49 AGCACAACCACGUAGGUGC  502 M1 CAGGCAAC S50-AS503- GCCUGGCACCUACGUGGUGUUGCTG   50 CAGCAACACCACGUAGGUG  503 M1 CCAGGCAA S51-AS504- AGGAGGAGACCCACCUCUCUCAGTC   51 GACUGAGAGAGGUGGGUCU  504 M1 CCUCCUUC S52-AS505- CCUGCAUGUCUUCCAUGGCUUUCTT   52 AAGAAAGCCAUGGAAGACA  505 M1 UGCAGGAU S53-AS506- UGCAUGUCUUCCAUGGCCUUCUUCC   53 GGAAGAAGGCCAUGGAAGA  506 M1 CAUGCAGG S54-AS507- ACCUGCUGGAGCUGGCCUUUAAGTT   54 AACUUAAAGGCCAGCUCCA  507 M1 GCAGGUCG S55-AS508- CUGCUGGAGCUGGCCUUGAAGUUGC   55 GCAACUUCAAGGCCAGCUC  508 M1 CAGCAGGU S56-AS509- UGCUGGAGCUGGCCUUGAAUUUGCC   56 GGCAAAUUCAAGGCCAGCU  509 M1 CCAGCAGG S57-AS510- UGGAGCUGGCCUUGAAGUUUCCCCA   57 UGGGGAAACUUCAAGGCCA  510 M1 GCUCCAGC S58-AS511- GGCCUUGAAGUUGCCCCAUUUCGAC   58 GUCGAAAUGGGGCAACUUC  511 M1 AAGGCCAG S59-AS512- GCCUUGAAGUUGCCCCAUGUCGACT   59 AGUCGACAUGGGGCAACUU  512 M1 CAAGGCCA S60-AS513- CCUUGAAGUUGCCCCAUGUUGACTA   60 UAGUCAACAUGGGGCAACU  513 M1 UCAAGGCC S61-AS514- CUUGAAGUUGCCCCAUGUCUACUAC   61 GUAGUAGACAUGGGGCAAC  514 M1 UUCAAGGC S62-AS515- ACUCCUCUGUCUUUGCCCAUAGCAT   62 AUGCUAUGGGCAAAGACAG  515 M1 AGGAGUCC S63-AS516- CUCCUCUGUCUUUGCCCAGAGCATC   63 GAUGCUCUGGGCAAAGACA  516 M1 GAGGAGUC S64-AS517- UCCUCUGUCUUUGCCCAGAUCAUCC   64 GGAUGAUCUGGGCAAAGAC  517 M1 AGAGGAGU S65-AS518- CCUCUGUCUUUGCCCAGAGUAUCCC   65 GGGAUACUCUGGGCAAAGA  518 M1 CAGAGGAG S66-AS519- UCUGUCUUUGCCCAGAGCAUCCCGT   66 ACGGGAUGCUCUGGGCAAA  519 M1 GACAGAGG S67-AS520- CUGUCUUUGCCCAGAGCAUUCCGTG   67 CACGGAAUGCUCUGGGCAA  520 M1 AGACAGAG S68-AS521- GUCUUUGCCCAGAGCAUCCUGUGGA   68 UCCACAGGAUGCUCUGGGC  521 M1 AAAGACAG S69-AS522- UCUUUGCCCAGAGCAUCCCUUGGAA   69 UUCCAAGGGAUGCUCUGGG  522 M1 CAAAGACA S70-AS523- UUUGCCCAGAGCAUCCCGUUGAACC   70 GGUUCAACGGGAUGCUCUG  523 M1 GGCAAAGA S71-AS524- AGAGCAUCCCGUGGAACCUUGAGCG   71 CGCUCAAGGUUCCACGGGA  524 M1 UGCUCUGG S72-AS525- GAGCAUCCCGUGGAACCUGUAGCGG   72 CCGCUACAGGUUCCACGGG  525 M1 AUGCUCUG S73-AS526- AGCAUCCCGUGGAACCUGGAGCGGA   73 UCCGCUCCAGGUUCCACGG  526 M1 GAUGCUCU S74-AS527- GCAUCCCGUGGAACCUGGAUCGGAT   74 AUCCGAUCCAGGUUCCACG  527 M1 GGAUGCUC S75-AS528- CAUCCCGUGGAACCUGGAGUGGATT   75 AAUCCACUCCAGGUUCCAC  528 M1 GGGAUGCU S76-AS529- AUCCCGUGGAACCUGGAGCUGAUTA   76 UAAUCAGCUCCAGGUUCCA  529 M1 CGGGAUGC S77-AS530- UCCCGUGGAACCUGGAGCGUAUUAC   77 GUAAUACGCUCCAGGUUCC  530 M1 ACGGGAUG S78-AS531- CCCGUGGAACCUGGAGCGGAUUACC   78 GGUAAUCCGCUCCAGGUUC  531 M1 CACGGGAU S79-AS532- CCGUGGAACCUGGAGCGGAUUACCC   79 GGGUAAUCCGCUCCAGGUU  532 M1 CCACGGGA S80-AS533- CUGGAGCGGAUUACCCCUCUACGGT   80 ACCGUAGAGGGGUAAUCCG  533 M1 CUCCAGGU S81-AS534- UGGAGCGGAUUACCCCUCCACGGTA   81 UACCGUGGAGGGGUAAUCC  534 M1 GCUCCAGG S82-AS535- GGAGCGGAUUACCCCUCCAUGGUAC   82 GUACCAUGGAGGGGUAAUC  535 M1 CGCUCCAG S83-AS536- GAGCGGAUUACCCCUCCACUGUACC   83 GGUACAGUGGAGGGGUAAU  536 M1 CCGCUCCA S84-AS537- AGCGGAUUACCCCUCCACGUUACCG   84 CGGUAACGUGGAGGGGUAA  537 M1 UCCGCUCC S85-AS538- CGGAUUACCCCUCCACGGUACCGGG   85 CCCGGUACCGUGGAGGGGU  538 M1 AAUCCGCU S86-AS539- GGAUUACCCCUCCACGGUAUCGGGC   86 GCCCGAUACCGUGGAGGGG  539 M1 UAAUCCGC S87-AS540- UCCACGGUACCGGGCGGAUUAAUAC   87 GUAUUAAUCCGCCCGGUAC  540 M1 CGUGGAGG S88-AS541- CGGAGGCAGCCUGGUGGAGUUGUAT   88 AUACAACUCCACCAGGCUG  541 M1 CCUCCGUC S89-AS542- AGACACCAGCAUACAGAGUUACCAC   89 GUGGUAACUCUGUAUGCUG  542 M1 GUGUCUAG S90-AS543- GCAUACAGAGUGACCACCGUGAAAT   90 AUUUCACGGUGGUCACUCU  543 M1 GUAUGCUG S91-AS544- CGAGAAUGUGCCCGAGGAGUACGGG   91 CCCGUACUCCUCGGGCACA  544 M1 UUCUCGAA S92-AS545- GAGAAUGUGCCCGAGGAGGACGGGA   92 UCCCGUCCUCCUCGGGCAC  545 M1 AUUCUCGA S93-AS546- AGAAUGUGCCCGAGGAGGAUGGGAC   93 GUCCCAUCCUCCUCGGGCA  546 M1 CAUUCUCG S94-AS547- GCAAGUGUGACAGUCAUGGUACCCA   94 UGGGUACCAUGACUGUCAC  547 M1 ACUUGCUG S95-AS548- CAAGUGUGACAGUCAUGGCACCCAC   95 GUGGGUGCCAUGACUGUCA  548 M1 CACUUGCU S96-AS549- AAGUGUGACAGUCAUGGCAUCCACC   96 GGUGGAUGCCAUGACUGUC  549 M1 ACACUUGC S97-AS550- CGCAGCCUGCGCGUGCUCAACUGCC   97 GGCAGUUGAGCACGCGCAG  550 M1 GCUGCGCA S98-AS551- GCAGCCUGCGCGUGCUCAAUUGCCA   98 UGGCAAUUGAGCACGCGCA  551 M1 GGCUGCGC S99-AS552- AGCCUGUGGGGCCACUGGUUGUGCT   99 AGCACAACCAGUGGCCCCA  552 M1 CAGGCUGG S100- CCUCUACUCCCCAGCCUCAUCUCCC  100 GGGAGAUGAGGCUGGGGAG  553 AS553-M1 UAGAGGCA S101- CAGCCUCAGCUCCCGAGGUUAUCAC  101 GUGAUAACCUCGGGAGCUG  554 AS554-M1 AGGCUGGG S102- GCCACCAAUGCCCAAGACCAGCCGG  102 CCGGCUGGUCUUGGGCAUU  555 AS555-M1 GGUGGCCC S103- AUGCCCAAGACCAGCCGGUUACCCT  103 AGGGUAACCGGCUGGUCUU  556 AS556-M1 GGGCAUUG S104- UGCCCAAGACCAGCCGGUGACCCTG  104 CAGGGUCACCGGCUGGUCU  557 AS557-M1 UGGGCAUU S105- GUCACAGAGUGGGACAUCAUAGGCT  105 AGCCUAUGAUGUCCCACUC  558 AS558-M1 UGUGACAC S106- GAGUGGGACAUCACAGGCUUCUGCC  106 GGCAGAAGCCUGUGAUGUC  559 AS559-M1 CCACUCUG S107- UGGGACAUCACAGGCUGCUUCCCAC  107 GUGGGAAGCAGCCUGUGAU  560 AS560-M1 GUCCCACU S108- GGGACAUCACAGGCUGCUGUCCACG  108 CGUGGACAGCAGCCUGUGA  561 AS561-M1 UGUCCCAC S109- CUCACCCUGGCCGAGUUGAUGCAGA  109 UCUGCAUCAACUCGGCCAG  562 AS562-M1 GGUGAGCU S110- ACCCUGGCCGAGUUGAGGCAGAGAC  110 GUCUCUGCCUCAACUCGGC  563 AS563-M1 CAGGGUGA S111- ACUUCUCUGCCAAAGAUGUUAUCAA  111 UUGAUAACAUCUUUGGCAG  564 AS564-M1 AGAAGUGG S112- CCCAUGGGGCAGGUUGGCAUCUGTT  112 AACAGAUGCCAACCUGCCC  565 AS565-M1 CAUGGGUG S113- UGGGGCAGGUUGGCAGCUGUUUUGC  113 GCAAAACAGCUGCCAACCU  566 AS566-M1 GCCCCAUG S114- CUGUUUUGCAGGACUGUAUUGUCAG  114 CUGACAAUACAGUCCUGCA  567 AS567-M1 AAACAGCU S115- UUUUGCAGGACUGUAUGGUUAGCAC  115 GUGCUAACCAUACAGUCCU  568 AS568-M1 GCAAAACA S116- CAGGACUGUAUGGUCAGCAUACUCG  116 CGAGUAUGCUGACCAUACA  569 AS569-M1 GUCCUGCA S117- GGACUGUAUGGUCAGCACAUUCGGG  117 CCCGAAUGUGCUGACCAUA  570 AS570-M1 CAGUCCUG S118- CGCUGCGCCCCAGAUGAGGAGCUGC  118 GCAGCUCCUCAUCUGGGGC  571 AS571-M1 GCAGCGGG S119- GCGCCCCAGAUGAGGAGCUUCUGAG  119 CUCAGAAGCUCCUCAUCUG  572 AS572-M1 GGGCGCAG S120- CCCCAGAUGAGGAGCUGCUUAGCTG  120 CAGCUAAGCAGCUCCUCAU  573 AS573-M1 CUGGGGCG S121- CCCAGAUGAGGAGCUGCUGAGCUGC  121 GCAGCUCAGCAGCUCCUCA  574 AS574-M1 UCUGGGGC S122- CCAGAUGAGGAGCUGCUGAUCUGCT  122 AGCAGAUCAGCAGCUCCUC  575 AS575-M1 AUCUGGGG S123- CGGCGGGGCGAGCGCAUGGAGGCCC  123 GGGCCUCCAUGCGCUCGCC  576 AS576-M1 CCGCCGCU S124- GGCGGGGCGAGCGCAUGGAUGCCCA  124 UGGGCAUCCAUGCGCUCGC  577 AS577-M1 CCCGCCGC S125- GGCGAGCGCAUGGAGGCCCAAGGGG  125 CCCCUUGGGCCUCCAUGCG  578 AS578-M1 CUCGCCCC S126- CUGGUCUGCCGGGCCCACAACGCTT  126 AAGCGUUGUGGGCCCGGCA  579 AS579-M1 GACCAGCU S127- UGCCUGCUACCCCAGGCCAACUGCA  127 UGCAGUUGGCCUGGGGUAG  580 AS580-M1 CAGGCAGC S128- GCCUGCUACCCCAGGCCAAUUGCAG  128 CUGCAAUUGGCCUGGGGUA  581 AS581-M1 GCAGGCAG S129- CCCAGGCCAACUGCAGCGUUCACAC  129 GUGUGAACGCUGCAGUUGG  582 AS582-M1 CCUGGGGU S130- GGCCCCUCAGGAGCAGGUGACCGTG  130 CACGGUCACCUGCUCCUGA  583 AS583-M1 GGGGCCGG S131- UGACCGUGGCCUGCGAGGAUGGCTG  131 CAGCCAUCCUCGCAGGCCA  584 AS584-M1 CGGUCACC S132- GCGAGGAGGGCUGGACCCUUACUGG  132 CCAGUAAGGGUCCAGCCCU  585 AS585-M1 CCUCGCAG S133- CGAGGAGGGCUGGACCCUGACUGGC  133 GCCAGUCAGGGUCCAGCCC  586 AS586-M1 UCCUCGCA S134- GGGCUGGACCCUGACUGGCUGCAGT  134 ACUGCAGCCAGUCAGGGUC  587 AS587-M1 CAGCCCUC S135- GGCUGGACCCUGACUGGCUUCAGTG  135 CACUGAAGCCAGUCAGGGU  588 AS588-M1 CCAGCCCU S136- UGGACCCUGACUGGCUGCAUUGCCC  136 GGGCAAUGCAGCCAGUCAG  589 AS589-M1 GGUCCAGC S137- GGCUGCAGUGCCCUCCCUGUGACCT  137 AGGUCACAGGGAGGGCACU  590 AS590-M1 GCAGCCAG S138- UCCCUGGGACCUCCCACGUUCUGGG  138 CCCAGAACGUGGGAGGUCC  591 AS591-M1 CAGGGAGG S139- CCCUGGGACCUCCCACGUCUUGGGG  139 CCCCAAGACGUGGGAGGUC  592 AS592-M1 CCAGGGAG S140- GGGCCUACGCCGUAGACAAUACGTG  140 CACGUAUUGUCUACGGCGU  593 AS593-M1 AGGCCCCC S141- GACGUCAGCACUACAGGCAUCACCA  141 UGGUGAUGCCUGUAGUGCU  594 AS594-M1 GACGUCCC S142- CAGCACUACAGGCAGCACCAGCGAA  142 UUCGCUGGUGCUGCCUGUA  595 AS595-M1 GUGCUGAC S143- AGCACUACAGGCAGCACCAUCGAAG  143 CUUCGAUGGUGCUGCCUGU  596 AS596-M1 AGUGCUGA S144- GCACUACAGGCAGCACCAGUGAAGG  144 CCUUCACUGGUGCUGCCUG  597 AS597-M1 UAGUGCUG S145- GGGGCCGUGACAGCCGUUGUCAUCT  145 AGAUGACAACGGCUGUCAC  598 AS598-M1 GGCCCCUU S146- GGAGCUCCAGUGACAGCCCUAUCCC  146 GGGAUAGGGCUGUCACUGG  599 AS599-M1 AGCUCCUG S147- AGGAUGGGUGUCUGGGGAGUGUCAA  147 UUGACACUCCCCAGACACC  600 AS600-M1 CAUCCUGG S148- UGGGUGUCUGGGGAGGGUCAAGGGC  148 GCCCUUGACCCUCCCCAGA  601 AS601-M1 CACCCAUC S149- GGGUGUCUGGGGAGGGUCAAGGGCT  149 AGCCCUUGACCCUCCCCAG  602 AS602-M1 CAACCCAU S150- GGUGUCUGGGGAGGGUCAAUGGCTG  150 CAGCCAUUGACCCUCCCCA  603 AS603-M1 AGCACCCA S151- AGGGUCAAGGGCUGGGGCUUAGCTT  151 AAGCUAAGCCCCAGCCCUU  604 AS604-M1 AGCCCUCC S152- GGGUCAAGGGCUGGGGCUGAGCUTT  152 AAAGCUCAGCCCCAGCCCU  605 AS605-M1 UGACCCUC S153- GACUUGUCCCUCUCUCAGCUCUCCA  153 UGGAGAGCUGAGAGAGGGA  606 AS606-M1 CAAGUCGG S154- ACUUGUCCCUCUCUCAGCCUUCCAT  154 AUGGAAGGCUGAGAGAGGG  607 AS607-M1 ACAAGUCG S155- CUUGUCCCUCUCUCAGCCCUCCATG  155 CAUGGAGGGCUGAGAGAGG  608 AS608-M1 GACAAGUC S156- UUGUCCCUCUCUCAGCCCUUCAUGG  156 CCAUGAAGGGCUGAGAGAG  609 AS609-M1 GGACAAGU S157- UCCCUCUCUCAGCCCUCCAUGGCCT  157 AGGCCAUGGAGGGCUGAGA  610 AS610-M1 GAGGGACA S158- UGGCCUGGCACGAGGGGAUUGGGAT  158 AUCCCAAUCCCCUCGUGCC  611 AS611-M1 AGGCCAUG S159- UGGCACGAGGGGAUGGGGAUGCUTC  159 GAAGCAUCCCCAUCCCCUC  612 AS612-M1 GUGCCAGG S160- CGAGGGGAUGGGGAUGCUUUCGCCT  160 AGGCGAAAGCAUCCCCAUC  613 AS613-M1 CCCUCGUG S161- GAGGGGAUGGGGAUGCUUCUGCCTT  161 AAGGCAGAAGCAUCCCCAU  614 AS614-M1 CCCCUCGU S162- GGGAUGGGGAUGCUUCCGCUUUUCC  162 GGAAAAGCGGAAGCAUCCC  615 AS615-M1 CAUCCCCU S163- AUGGGGAUGCUUCCGCCUUUCCGGG  163 CCCGGAAAGGCGGAAGCAU  616 AS616-M1 CCCCAUCC S164- UGGGGAUGCUUCCGCCUUUUCGGGG  164 CCCCGAAAAGGCGGAAGCA  617 AS617-M1 UCCCCAUC S165- GGGGAUGCUUCCGCCUUUCUGGGGC  165 GCCCCAGAAAGGCGGAAGC  618 AS618-M1 AUCCCCAU S166- GGGAUGCUUCCGCCUUUCCUGGGCT  166 AGCCCAGGAAAGGCGGAAG  619 AS619-M1 CAUCCCCA S167- CCCUUGAGUGGGGCAGCCUUCUUGC  167 GCAAGAAGGCUGCCCCACU  620 AS620-M1 CAAGGGCC S168- UGAGUGGGGCAGCCUCCUUUCCUGG  168 CCAGGAAAGGAGGCUGCCC  621 AS621-M1 CACUCAAG S169- GGGGCAGCCUCCUUGCCUGUAACTC  169 GAGUUACAGGCAAGGAGGC  622 AS622-M1 UGCCCCAC S170- GGCAGCCUCCUUGCCUGGAACUCAC  170 GUGAGUUCCAGGCAAGGAG  623 AS623-M1 GCUGCCCC S171- GCAGCCUCCUUGCCUGGAAUUCACT  171 AGUGAAUUCCAGGCAAGGA  624 AS624-M1 GGCUGCCC S172- AGCCUCCUUGCCUGGAACUUACUCA  172 UGAGUAAGUUCCAGGCAAG  625 AS625-M1 GAGGCUGC S173- GCCUCCUUGCCUGGAACUCACUCAC  173 GUGAGUGAGUUCCAGGCAA  626 AS626-M1 GGAGGCUG S174- CCUCCUUGCCUGGAACUCAUUCACT  174 AGUGAAUGAGUUCCAGGCA  627 AS627-M1 AGGAGGCU S175- CUCCUUGCCUGGAACUCACUCACTC  175 GAGUGAGUGAGUUCCAGGC  628 AS628-M1 AAGGAGGC S176- UCCUUGCCUGGAACUCACUUACUCT  176 AGAGUAAGUGAGUUCCAGG  629 AS629-M1 CAAGGAGG S177- CCUUGCCUGGAACUCACUCACUCTG  177 CAGAGUGAGUGAGUUCCAG  630 AS630-M1 GCAAGGAG S178- CUUGCCUGGAACUCACUCAUUCUGG  178 CCAGAAUGAGUGAGUUCCA  631 AS631-M1 GGCAAGGA S179- UUGCCUGGAACUCACUCACUCUGGG  179 CCCAGAGUGAGUGAGUUCC  632 AS632-M1 AGGCAAGG S180- UGCCUGGAACUCACUCACUUUGGGT  180 ACCCAAAGUGAGUGAGUUC  633 AS633-M1 CAGGCAAG S181- UCUGGGUGCCUCCUCCCCAUGUGGA  181 UCCACAUGGGGAGGAGGCA  634 AS634-M1 CCCAGAGU S182- CCCAGGUGGAGGUGCCAGGAAGCTC  182 GAGCUUCCUGGCACCUCCA  635 AS635-M1 CCUGGGGA S183- CCAGGAAGCUCCCUCCCUCACUGTG  183 CACAGUGAGGGAGGGAGCU  636 AS636-M1 UCCUGGCA S184- GGAAGCUCCCUCCCUCACUUUGGGG  184 CCCCAAAGUGAGGGAGGGA  637 AS637-M1 GCUUCCUG S185- AGCUCCCUCCCUCACUGUGUGGCAT  185 AUGCCACACAGUGAGGGAG  638 AS638-M1 GGAGCUUC S186- GCUCCCUCCCUCACUGUGGUGCATT  186 AAUGCACCACAGUGAGGGA  639 AS639-M1 GGGAGCUU S187- GGGGCAUUUCACCAUUCAAACAGGT  187 ACCUGUUUGAAUGGUGAAA  640 AS640-M1 UGCCCCAC S188- GGGCAUUUCACCAUUCAAAUAGGTC  188 GACCUAUUUGAAUGGUGAA  641 AS641-M1 AUGCCCCA S189- CACCAUUCAAACAGGUCGAUCUGTG  189 CACAGAUCGACCUGUUUGA  642 AS642-M1 AUGGUGAA S190- ACCAUUCAAACAGGUCGAGUUGUGC  190 GCACAACUCGACCUGUUUG  643 AS643-M1 AAUGGUGA S191- UGCUCGGGUGCUGCCAGCUUCUCCC  191 GGGAGAAGCUGGCAGCACC  644 AS644-M1 CGAGCACA S192- CGGGUGCUGCCAGCUGCUCUCAATG  192 CAUUGAGAGCAGCUGGCAG  645 AS645-M1 CACCCGAG S193- GGGUGCUGCCAGCUGCUCCUAAUGT  193 ACAUUAGGAGCAGCUGGCA  646 AS646-M1 GCACCCGA S194- GCCAGCUGCUCCCAAUGUGUCGATG  194 CAUCGACACAUUGGGAGCA  647 AS647-M1 GCUGGCAG S195- CCAGCUGCUCCCAAUGUGCUGAUGT  195 ACAUCAGCACAUUGGGAGC  648 AS648-M1 AGCUGGCA S196- UGCCGAUGUCCGUGGGCAGAAUGAC  196 GUCAUUCUGCCCACGGACA  649 AS649-M1 UCGGCACA S197- GCAGAAUGACUUUUAUUGAUCUCTT  197 AAGAGAUCAAUAAAAGUCA  650 AS650-M1 UUCUGCCC S198- CAGAAUGACUUUUAUUGAGUUCUTG  198 CAAGAACUCAAUAAAAGUC  651 AS651-M1 AUUCUGCC S199- AGAAUGACUUUUAUUGAGCUCUUGT  199 ACAAGAGCUCAAUAAAAGU  652 AS652-M1 CAUUCUGC S200- GAAUGACUUUUAUUGAGCUUUUGTT  200 AACAAAAGCUCAAUAAAAG  653 AS653-M1 UCAUUCUG S201- AAUGACUUUUAUUGAGCUCUUGUTC  201 GAACAAGAGCUCAAUAAAA  654 AS654-M1 GUCAUUCU S202- AUGACUUUUAUUGAGCUCUUGUUCC  202 GGAACAAGAGCUCAAUAAA  655 AS655-M1 AGUCAUUC S203- UGACUUUUAUUGAGCUCUUUUUCCG  203 CGGAAAAAGAGCUCAAUAA  656 AS656-M1 AAGUCAUU S204- CUUGUUCCGUGCCAGGCAUUCAATC  204 GAUUGAAUGCCUGGCACGG  657 AS657-M1 AACAAGAG S205- CCAGGCAUUCAAUCCUCAGUUCUCC  205 GGAGAACUGAGGAUUGAAU  658 AS658-M1 GCCUGGCA S206- CAUUCAAUCCUCAGGUCUCUACCAA  206 UUGGUAGAGACCUGAGGAU  659 AS659-M1 UGAAUGCC S207- AUUCAAUCCUCAGGUCUCCACCAAG  207 CUUGGUGGAGACCUGAGGA  660 AS660-M1 UUGAAUGC S208- UUCAAUCCUCAGGUCUCCAUCAAGG  208 CCUUGAUGGAGACCUGAGG  661 AS661-M1 AUUGAAUG S209- CCUCAGGUCUCCACCAAGGAGGCAG  209 CUGCCUCCUUGGUGGAGAC  662 AS662-M1 CUGAGGAU S210- CUCAGGUCUCCACCAAGGAUGCAGG  210 CCUGCAUCCUUGGUGGAGA  663 AS663-M1 CCUGAGGA S211- GCGGUAGGGGCUGCAGGGAUAAACA  211 UGUUUAUCCCUGCAGCCCC  664 AS664-M1 UACCGCCC S212- CGGUAGGGGCUGCAGGGACAAACAT  212 AUGUUUGUCCCUGCAGCCC  665 AS665-M1 CUACCGCC S213- GGUAGGGGCUGCAGGGACAAACATC  213 GAUGUUUGUCCCUGCAGCC  666 AS666-M1 CCUACCGC S214- UAGGGGCUGCAGGGACAAAUAUCGT  214 ACGAUAUUUGUCCCUGCAG  667 AS667-M1 CCCCUACC S215- AGGGGCUGCAGGGACAAACAUCGTT  215 AACGAUGUUUGUCCCUGCA  668 AS668-M1 GCCCCUAC S216- GGGGCUGCAGGGACAAACAUCGUTG  216 CAACGAUGUUUGUCCCUGC  669 AS669-M1 AGCCCCUA S217- GGGCUGCAGGGACAAACAUUGUUGG  217 CCAACAAUGUUUGUCCCUG  670 AS670-M1 CAGCCCCU S218- GGCUGCAGGGACAAACAUCUUUGGG  218 CCCAAAGAUGUUUGUCCCU  671 AS671-M1 GCAGCCCC S219- GGGGUGAGUGUGAAAGGUGUUGATG  219 CAUCAACACCUUUCACACU  672 AS672-M1 CACCCCCC S220- GGGUGAGUGUGAAAGGUGCUGAUGG  220 CCAUCAGCACCUUUCACAC  673 AS673-M1 UCACCCCC S221- GGUGAGUGUGAAAGGUGCUUAUGGC  221 GCCAUAAGCACCUUUCACA  674 AS674-M1 CUCACCCC S222- GUGAGUGUGAAAGGUGCUGAUGGCC  222 GGCCAUCAGCACCUUUCAC  675 AS675-M1 ACUCACCC S223- UGAGUGUGAAAGGUGCUGAUGGCCC  223 GGGCCAUCAGCACCUUUCA  676 AS676-M1 CACUCACC S224- GAGUGUGAAAGGUGCUGAUUGCCCT  224 AGGGCAAUCAGCACCUUUC  677 AS677-M1 ACACUCAC S225- AGUGUGAAAGGUGCUGAUGUCCCTC  225 GAGGGACAUCAGCACCUUU  678 AS678-M1 CACACUCA S226- GUGUGAAAGGUGCUGAUGGUCCUCA  226 UGAGGACCAUCAGCACCUU  679 AS679-M1 UCACACUC S227- UGUGAAAGGUGCUGAUGGCUCUCAT  227 AUGAGAGCCAUCAGCACCU  680 AS680-M1 UUCACACU S228- GUGAAAGGUGCUGAUGGCCUUCATC  228 GAUGAAGGCCAUCAGCACC  681 AS681-M1 UUUCACAC S229- UGAAAGGUGCUGAUGGCCCUCAUCT  229 AGAUGAGGGCCAUCAGCAC  682 AS682-M1 CUUUCACA S230- GAAAGGUGCUGAUGGCCCUUAUCTC  230 GAGAUAAGGGCCAUCAGCA  683 AS683-M1 CCUUUCAC S231- CUCAUCUCCAGCUAACUGUUGAGAA  231 UUCUCAACAGUUAGCUGGA  684 AS684-M1 GAUGAGGG S232- CCAGCUAACUGUGGAGAAGUCCCTG  232 CAGGGACUUCUCCACAGUU  685 AS685-M1 AGCUGGAG S233- CAGCUAACUGUGGAGAAGCUCCUGG  233 CCAGGAGCUUCUCCACAGU  686 AS686-M1 UAGCUGGA S234- AGCUAACUGUGGAGAAGCCUCUGGG  234 CCCAGAGGCUUCUCCACAG  687 AS687-M1 UUAGCUGG S235- GCUAACUGUGGAGAAGCCCUUGGGG  235 CCCCAAGGGCUUCUCCACA  688 AS688-M1 GUUAGCUG S236- GGGCUCCCUGAUUAAUGGAUGCUTA  236 UAAGCAUCCAUUAAUCAGG  689 AS689-M1 GAGCCCCC S237- AUGGAGGCUUAGCUUUCUGUAUGGC  237 GCCAUACAGAAAGCUAAGC  690 AS690-M1 CUCCAUUA S238- UGGAGGCUUAGCUUUCUGGAUGGCA  238 UGCCAUCCAGAAAGCUAAG  691 AS691-M1 CCUCCAUU S239- GGAGGCUUAGCUUUCUGGAUGGCAT  239 AUGCCAUCCAGAAAGCUAA  692 AS692-M1 GCCUCCAU S240- GAGGCUUAGCUUUCUGGAUUGCATC  240 GAUGCAAUCCAGAAAGCUA  693 AS693-M1 AGCCUCCA S241- AGGCUUAGCUUUCUGGAUGUCAUCT  241 AGAUGACAUCCAGAAAGCU  694 AS694-M1 AAGCCUCC S242- GGCUUAGCUUUCUGGAUGGUAUCTA  242 UAGAUACCAUCCAGAAAGC  695 AS695-M1 UAAGCCUC S243- GCUUAGCUUUCUGGAUGGCAUCUAG  243 CUAGAUGCCAUCCAGAAAG  696 AS696-M1 CUAAGCCU S244- GACAGGUGCGCCCCUGGUGUUCACA  244 UGUGAACACCAGGGGCGCA  697 AS697-M1 CCUGUCUC S245- GCGCCCCUGGUGGUCACAGUCUGTG  245 CACAGACUGUGACCACCAG  698 AS698-M1 GGGCGCAC S246- CCCCUGGUGGUCACAGGCUUUGCCT  246 AGGCAAAGCCUGUGACCAC  699 AS699-M1 CAGGGGCG S247- CCCUGGUGGUCACAGGCUGUGCCTT  247 AAGGCACAGCCUGUGACCA  700 AS700-M1 CCAGGGGC S248- GUGGUCACAGGCUGUGCCUUGGUTT  248 AAACCAAGGCACAGCCUGU  701 AS701-M1 GACCACCA S249- UGGUCACAGGCUGUGCCUUUGUUTC  249 GAAACAAAGGCACAGCCUG  702 AS702-M1 UGACCACC S250- GGUCACAGGCUGUGCCUUGUUUUCC  250 GGAAAACAAGGCACAGCCU  703 AS703-M1 GUGACCAC S251- GUCACAGGCUGUGCCUUGGUUUCCT  251 AGGAAACCAAGGCACAGCC  704 AS704-M1 UGUGACCA S252- GGCUGUGCCUUGGUUUCCUUAGCCA  252 UGGCUAAGGAAACCAAGGC  705 AS705-M1 ACAGCCUG S253- GCUGUGCCUUGGUUUCCUGAGCCAC  253 GUGGCUCAGGAAACCAAGG  706 AS706-M1 CACAGCCU S254- CUGUGCCUUGGUUUCCUGAUCCACC  254 GGUGGAUCAGGAAACCAAG  707 AS707-M1 GCACAGCC S255- UGUGCCUUGGUUUCCUGAGUCACCT  255 AGGUGACUCAGGAAACCAA  708 AS708-M1 GGCACAGC S256- GUGCCUUGGUUUCCUGAGCUACCTT  256 AAGGUAGCUCAGGAAACCA  709 AS709-M1 AGGCACAG S257- UGCCUUGGUUUCCUGAGCCACCUTT  257 AAAGGUGGCUCAGGAAACC  710 AS710-M1 AAGGCACA S258- GCCUUGGUUUCCUGAGCCAUCUUTA  258 UAAAGAUGGCUCAGGAAAC  711 AS711-M1 CAAGGCAC S259- CCUUGGUUUCCUGAGCCACUUUUAC  259 GUAAAAGUGGCUCAGGAAA  712 AS712-M1 CCAAGGCA S260- CUUGGUUUCCUGAGCCACCUUUACT  260 AGUAAAGGUGGCUCAGGAA  713 AS713-M1 ACCAAGGC S261- UUGGUUUCCUGAGCCACCUUUACTC  261 GAGUAAAGGUGGCUCAGGA  714 AS714-M1 AACCAAGG S262- UGGUUUCCUGAGCCACCUUUACUCT  262 AGAGUAAAGGUGGCUCAGG  715 AS715-M1 AAACCAAG S263- GGUUUCCUGAGCCACCUUUACUCTG  263 CAGAGUAAAGGUGGCUCAG  716 AS716-M1 GAAACCAA S264- GUUUCCUGAGCCACCUUUAUUCUGC  264 GCAGAAUAAAGGUGGCUCA  717 AS717-M1 GGAAACCA S265- CUGAGCCACCUUUACUCUGUUCUAT  265 AUAGAACAGAGUAAAGGUG  718 AS718-M1 GCUCAGGA S266- CCAGGCUGUGCUAGCAACAUCCAAA  266 UUUGGAUGUUGCUAGCACA  719 AS719-M1 GCCUGGCA S267- CUGCGGGGAGCCAUCACCUAGGACT  267 AGUCCUAGGUGAUGGCUCC  720 AS720-M1 CCGCAGGC S268- UGCGGGGAGCCAUCACCUAUGACTG  268 CAGUCAUAGGUGAUGGCUC  721 AS721-M1 CCCGCAGG S269- GCGGGGAGCCAUCACCUAGUACUGA  269 UCAGUACUAGGUGAUGGCU  722 AS722-M1 CCCCGCAG S270- CGGGGAGCCAUCACCUAGGACUGAC  270 GUCAGUCCUAGGUGAUGGC  723 AS723-M1 UCCCCGCA S271- GGGGAGCCAUCACCUAGGAUUGACT  271 AGUCAAUCCUAGGUGAUGG  724 AS724-M1 CUCCCCGC S272- GCCAUCACCUAGGACUGACUCGGCA  272 UGCCGAGUCAGUCCUAGGU  725 AS725-M1 GAUGGCUC S273- CCAUCACCUAGGACUGACUUGGCAG  273 CUGCCAAGUCAGUCCUAGG  726 AS726-M1 UGAUGGCU S274- CAUCACCUAGGACUGACUCUGCAGT  274 ACUGCAGAGUCAGUCCUAG  727 AS727-M1 GUGAUGGC S275- CUAGGACUGACUCGGCAGUUUGCAG  275 CUGCAAACUGCCGAGUCAG  728 AS728-M1 UCCUAGGU S276- UGACUCGGCAGUGUGCAGUUGUGCA  276 UGCACAACUGCACACUGCC  729 AS729-M1 GAGUCAGU S277- GACUCGGCAGUGUGCAGUGUUGCAT  277 AUGCAACACUGCACACUGC  730 AS730-M1 CGAGUCAG S278- CUCGGCAGUGUGCAGUGGUUCAUGC  278 GCAUGAACCACUGCACACU  731 AS731-M1 GCCGAGUC S279- UCGGCAGUGUGCAGUGGUGUAUGCA  279 UGCAUACACCACUGCACAC  732 AS732-M1 UGCCGAGU S280- CGGCAGUGUGCAGUGGUGCAUGCAC  280 GUGCAUGCACCACUGCACA  733 AS733-M1 CUGCCGAG S281- GUGUGCAGUGGUGCAUGCAUUGUCT  281 AGACAAUGCAUGCACCACU  734 AS734-M1 GCACACUG S282- UGUGCAGUGGUGCAUGCACUGUCTC  282 GAGACAGUGCAUGCACCAC  735 AS735-M1 UGCACACU S283- GUGCAGUGGUGCAUGCACUUUCUCA  283 UGAGAAAGUGCAUGCACCA  736 AS736-M1 CUGCACAC S284- UGCAGUGGUGCAUGCACUGUCUCAG  284 CUGAGACAGUGCAUGCACC  737 AS737-M1 ACUGCACA S285- GCAGUGGUGCAUGCACUGUUUCAGC  285 GCUGAAACAGUGCAUGCAC  738 AS738-M1 CACUGCAC S286- CAGUGGUGCAUGCACUGUCUCAGCC  286 GGCUGAGACAGUGCAUGCA  739 AS739-M1 CCACUGCA S287- AGUGGUGCAUGCACUGUCUUAGCCA  287 UGGCUAAGACAGUGCAUGC  740 AS740-M1 ACCACUGC S288- UGCAUGCACUGUCUCAGCCAACCCG  288 CGGGUUGGCUGAGACAGUG  741 AS741-M1 CAUGCACC S289- GCAUGCACUGUCUCAGCCAACCCGC  289 GCGGGUUGGCUGAGACAGU  742 AS742-M1 GCAUGCAC S290- CAUUCGCACCCCUACUUCAUAGAGG  290 CCUCUAUGAAGUAGGGGUG  743 AS743-M1 CGAAUGUG S291- AUUCGCACCCCUACUUCACAGAGGA  291 UCCUCUGUGAAGUAGGGGU  744 AS744-M1 GCGAAUGU S292- UUCGCACCCCUACUUCACAUAGGAA  292 UUCCUAUGUGAAGUAGGGG  745 AS745-M1 UGCGAAUG S293- UCGCACCCCUACUUCACAGAGGAAG  293 CUUCCUCUGUGAAGUAGGG  746 AS746-M1 GUGCGAAU S294- CGCACCCCUACUUCACAGAUGAAGA  294 UCUUCAUCUGUGAAGUAGG  747 AS747-M1 GGUGCGAA S295- GCACCCCUACUUCACAGAGUAAGAA  295 UUCUUACUCUGUGAAGUAG  748 AS748-M1 GGGUGCGA S296- CACCCCUACUUCACAGAGGAAGAAA  296 UUUCUUCCUCUGUGAAGUA  749 AS749-M1 GGGGUGCG S297- ACCCCUACUUCACAGAGGAAGAAAC  297 GUUUCUUCCUCUGUGAAGU  750 AS750-M1 AGGGGUGC S298- CCCCUACUUCACAGAGGAAUAAACC  298 GGUUUAUUCCUCUGUGAAG  751 AS751-M1 UAGGGGUG S299- CCCUACUUCACAGAGGAAGAAACCT  299 AGGUUUCUUCCUCUGUGAA  752 AS752-M1 GUAGGGGU S300- CUUCACAGAGGAAGAAACCUGGAAC  300 GUUCCAGGUUUCUUCCUCU  753 AS753-M1 GUGAAGUA S301- UUCACAGAGGAAGAAACCUUGAACC  301 GGUUCAAGGUUUCUUCCUC  754 AS754-M1 UGUGAAGU S302- UCACAGAGGAAGAAACCUGUAACCA  302 UGGUUACAGGUUUCUUCCU  755 AS755-M1 CUGUGAAG S303- CACAGAGGAAGAAACCUGGAACCAG  303 CUGGUUCCAGGUUUCUUCC  756 AS756-M1 UCUGUGAA S304- ACAGAGGAAGAAACCUGGAACCAGA  304 UCUGGUUCCAGGUUUCUUC  757 AS757-M1 CUCUGUGA S305- CAGAGGAAGAAACCUGGAAUCAGAG  305 CUCUGAUUCCAGGUUUCUU  758 AS758-M1 CCUCUGUG S306- AGAGGAAGAAACCUGGAACUAGAGG  306 CCUCUAGUUCCAGGUUUCU  759 AS759-M1 UCCUCUGU S307- GAGGAAGAAACCUGGAACCAGAGGG  307 CCCUCUGGUUCCAGGUUUC  760 AS760-M1 UUCCUCUG S308- AGGAAGAAACCUGGAACCAUAGGGG  308 CCCCUAUGGUUCCAGGUUU  761 AS761-M1 CUUCCUCU S309- GCAGAUUGGGCUGGCUCUGAAGCCA  309 UGGCUUCAGAGCCAGCCCA  762 AS762-M1 AUCUGCGU S310- CAGAUUGGGCUGGCUCUGAAGCCAA  310 UUGGCUUCAGAGCCAGCCC  763 AS763-M1 AAUCUGCG S311- AGAUUGGGCUGGCUCUGAAUCCAAG  311 CUUGGAUUCAGAGCCAGCC  764 AS764-M1 CAAUCUGC S312- UGGGCUGGCUCUGAAGCCAAGCCTC  312 GAGGCUUGGCUUCAGAGCC  765 AS765-M1 AGCCCAAU S313- GGGCUGGCUCUGAAGCCAAUCCUCT  313 AGAGGAUUGGCUUCAGAGC  766 AS766-M1 CAGCCCAA S314- GAAGCCAAGCCUCUUCUUAUUUCAC  314 GUGAAAUAAGAAGAGGCUU  767 AS767-M1 GGCUUCAG S315- AAGCCUCUUCUUACUUCACUCGGCT  315 AGCCGAGUGAAGUAAGAAG  768 AS768-M1 AGGCUUGG S316- AGCCUCUUCUUACUUCACCUGGCTG  316 CAGCCAGGUGAAGUAAGAA  769 AS769-M1 GAGGCUUG S317- GCCUCUUCUUACUUCACCCUGCUGG  317 CCAGCAGGGUGAAGUAAGA  770 AS770-M1 AGAGGCUU S318- CCCGGCUGGGCUCCUCAUUUUUACG  318 CGUAAAAAUGAGGAGCCCA  771 AS771-M1 GCCGGGUG S319- CCGGCUGGGCUCCUCAUUUUUACGG  319 CCGUAAAAAUGAGGAGCCC  772 AS772-M1 AGCCGGGU S320- CGGCUGGGCUCCUCAUUUUUACGGG  320 CCCGUAAAAAUGAGGAGCC  773 AS773-M1 CAGCCGGG S321- GGCUGGGCUCCUCAUUUUUACGGGT  321 ACCCGUAAAAAUGAGGAGC  774 AS774-M1 CCAGCCGG S322- GCUGGGCUCCUCAUUUUUAUGGGTA  322 UACCCAUAAAAAUGAGGAG  775 AS775-M1 CCCAGCCG S323- ACGGGUAACAGUGAGGCUGUGAAGG  323 CCUUCACAGCCUCACUGUU  776 AS776-M1 ACCCGUAA S324- AGCUCGGUGAGUGAUGGCAUAACGA  324 UCGUUAUGCCAUCACUCAC  777 AS777-M1 CGAGCUUC S325- GCUCGGUGAGUGAUGGCAGAACGAT  325 AUCGUUCUGCCAUCACUCA  778 AS778-M1 CCGAGCUU S326- CUCGGUGAGUGAUGGCAGAACGATG  326 CAUCGUUCUGCCAUCACUC  779 AS779-M1 ACCGAGCU S327- UCGGUGAGUGAUGGCAGAAUGAUGC  327 GCAUCAUUCUGCCAUCACU  780 AS780-M1 CACCGAGC S328- CGGUGAGUGAUGGCAGAACUAUGCC  328 GGCAUAGUUCUGCCAUCAC  781 AS781-M1 UCACCGAG S329- AUGCCUGCAGGCAUGGAACUUUUTC  329 GAAAAAGUUCCAUGCCUGC  782 AS782-M1 AGGCAUCG S330- UGCCUGCAGGCAUGGAACUUUUUCC  330 GGAAAAAGUUCCAUGCCUG  783 AS783-M1 CAGGCAUC S331- GCCUGCAGGCAUGGAACUUUUUCCG  331 CGGAAAAAGUUCCAUGCCU  784 AS784-M1 GCAGGCAU S332- CCUGCAGGCAUGGAACUUUUUCCGT  332 ACGGAAAAAGUUCCAUGCC  785 AS785-M1 UGCAGGCA S333- CUGCAGGCAUGGAACUUUUUCCGTT  333 AACGGAAAAAGUUCCAUGC  786 AS786-M1 CUGCAGGC S334- AUGGAACUUUUUCCGUUAUUACCCA  334 UGGGUAAUAACGGAAAAAG  787 AS787-M1 UUCCAUGC S335- UUUUUCCGUUAUCACCCAGUCCUGA  335 UCAGGACUGGGUGAUAACG  788 AS788-M1 GAAAAAGU S336- UUUUCCGUUAUCACCCAGGUCUGAT  336 AUCAGACCUGGGUGAUAAC  789 AS789-M1 GGAAAAAG S337- UUUCCGUUAUCACCCAGGCUUGATT  337 AAUCAAGCCUGGGUGAUAA  790 AS790-M1 CGGAAAAA S338- UUCCGUUAUCACCCAGGCCUGAUTC  338 GAAUCAGGCCUGGGUGAUA  791 AS791-M1 ACGGAAAA S339- UCCGUUAUCACCCAGGCCUUAUUCA  339 UGAAUAAGGCCUGGGUGAU  792 AS792-M1 AACGGAAA S340- CCGUUAUCACCCAGGCCUGAUUCAC  340 GUGAAUCAGGCCUGGGUGA  793 AS793-M1 UAACGGAA S341- CGUUAUCACCCAGGCCUGAUUCACT  341 AGUGAAUCAGGCCUGGGUG  794 AS794-M1 AUAACGGA S342- CACCCAGGCCUGAUUCACUUGCCTG  342 CAGGCAAGUGAAUCAGGCC  795 AS795-M1 UGGGUGAU S343- ACCCAGGCCUGAUUCACUGUCCUGG  343 CCAGGACAGUGAAUCAGGC  796 AS796-M1 CUGGGUGA S344- UGGCCUGGCGGAGAUGCUUUUAAGG  344 CCUUAAAAGCAUCUCCGCC  797 AS797-M1 AGGCCAGU S345- GGCCUGGCGGAGAUGCUUCUAAGGC  345 GCCUUAGAAGCAUCUCCGC  798 AS798-M1 CAGGCCAG S346- GCCUGGCGGAGAUGCUUCUAAGGCA  346 UGCCUUAGAAGCAUCUCCG  799 AS799-M1 CCAGGCCA S347- CCUGGCGGAGAUGCUUCUAAGGCAT  347 AUGCCUUAGAAGCAUCUCC  800 AS800-M1 GCCAGGCC S348- CUGGCGGAGAUGCUUCUAAUGCATG  348 CAUGCAUUAGAAGCAUCUC  801 AS801-M1 CGCCAGGC S349- UGGCGGAGAUGCUUCUAAGUCAUGG  349 CCAUGACUUAGAAGCAUCU  802 AS802-M1 CCGCCAGG S350- GGCGGAGAUGCUUCUAAGGUAUGGT  350 ACCAUACCUUAGAAGCAUC  803 AS803-M1 UCCGCCAG S351- GCGGAGAUGCUUCUAAGGCAUGGTC  351 GACCAUGCCUUAGAAGCAU  804 AS804-M1 CUCCGCCA S352- CGGAGAUGCUUCUAAGGCAUGGUCG  352 CGACCAUGCCUUAGAAGCA  805 AS805-M1 UCUCCGCC S353- GGAGAUGCUUCUAAGGCAUUGUCGG  353 CCGACAAUGCCUUAGAAGC  806 AS806-M1 AUCUCCGC S354- GAGAUGCUUCUAAGGCAUGUUCGGG  354 CCCGAACAUGCCUUAGAAG  807 AS807-M1 CAUCUCCG S355- GGAGAGGGCCAACAACUGUUCCUCC  355 GGAGGAACAGUUGUUGGCC  808 AS808-M1 CUCUCCCC S356- GCCAACAACUGUCCCUCCUUGAGCA  356 UGCUCAAGGAGGGACAGUU  809 AS809-M1 GUUGGCCC S357- CCAACAACUGUCCCUCCUUUAGCAC  357 GUGCUAAAGGAGGGACAGU  810 AS810-M1 UGUUGGCC S358- UUGAGCACCAGCCCCACCCAAGCAA  358 UUGCUUGGGUGGGGCUGGU  811 AS811-M1 GCUCAAGG S359- UGAGCACCAGCCCCACCCAAGCAAG  359 CUUGCUUGGGUGGGGCUGG  812 AS812-M1 UGCUCAAG S360- GAGCACCAGCCCCACCCAAUCAAGC  360 GCUUGAUUGGGUGGGGCUG  813 AS813-M1 GUGCUCAA S361- AGCACCAGCCCCACCCAAGUAAGCA  361 UGCUUACUUGGGUGGGGCU  814 AS814-M1 GGUGCUCA S362- ACCCAAGCAAGCAGACAUUUAUCTT  362 AAGAUAAAUGUCUGCUUGC  815 AS815-M1 UUGGGUGG S363- CCCAAGCAAGCAGACAUUUAUCUTT  363 AAAGAUAAAUGUCUGCUUG  816 AS816-M1 CUUGGGUG S364- CCAAGCAAGCAGACAUUUAUCUUTT  364 AAAAGAUAAAUGUCUGCUU  817 AS817-M1 GCUUGGGU S365- CAAGCAAGCAGACAUUUAUUUUUTG  365 CAAAAAAUAAAUGUCUGCU  818 AS818-M1 UGCUUGGG S366- AAGCAAGCAGACAUUUAUCUUUUGG  366 CCAAAAGAUAAAUGUCUGC  819 AS819-M1 UUGCUUGG S367- AGCAAGCAGACAUUUAUCUUUUGGG  367 CCCAAAAGAUAAAUGUCUG  820 AS820-M1 CUUGCUUG S368- GCAAGCAGACAUUUAUCUUUUGGGT  368 ACCCAAAAGAUAAAUGUCU  821 AS821-M1 GCUUGCUU S369- AAGCAGACAUUUAUCUUUUUGGUCT  369 AGACCAAAAAGAUAAAUGU  822 AS822-M1 CUGCUUGC S370- AGCAGACAUUUAUCUUUUGUGUCTG  370 CAGACACAAAAGAUAAAUG  823 AS823-M1 UCUGCUUG S371- GCAGACAUUUAUCUUUUGGUUCUGT  371 ACAGAACCAAAAGAUAAAU  824 AS824-M1 GUCUGCUU S372- UGUUGCCUUUUUACAGCCAACUUTT  372 AAAAGUUGGCUGUAAAAAG  825 AS825-M1 GCAACAGA S373- GUUGCCUUUUUACAGCCAAUUUUTC  373 GAAAAAUUGGCUGUAAAAA  826 AS826-M1 GGCAACAG S374- UUUACAGCCAACUUUUCUAUACCTG  374 CAGGUAUAGAAAAGUUGGC  827 AS827-M1 UGUAAAAA S375- UUACAGCCAACUUUUCUAGACCUGT  375 ACAGGUCUAGAAAAGUUGG  828 AS828-M1 CUGUAAAA S376- UUUUCUAGACCUGUUUUGCUUUUGT  376 ACAAAAGCAAAACAGGUCU  829 AS829-M1 AGAAAAGU S377- UUUCUAGACCUGUUUUGCUUUUGTA  377 UACAAAAGCAAAACAGGUC  830 AS830-M1 UAGAAAAG S378- UUCUAGACCUGUUUUGCUUUUGUAA  378 UUACAAAAGCAAAACAGGU  831 AS831-M1 CUAGAAAA S379- UCUAGACCUGUUUUGCUUUUGUAAC  379 GUUACAAAAGCAAAACAGG  832 AS832-M1 UCUAGAAA S380- CUAGACCUGUUUUGCUUUUUUAACT  380 AGUUAAAAAAGCAAAACAG  833 AS833-M1 GUCUAGAA S381- UAGACCUGUUUUGCUUUUGUAACTT  381 AAGUUACAAAAGCAAAACA  834 AS834-M1 GGUCUAGA S382- AGACCUGUUUUGCUUUUGUAACUTG  382 CAAGUUACAAAAGCAAAAC  835 AS835-M1 AGGUCUAG S383- GACCUGUUUUGCUUUUGUAACUUGA  383 UCAAGUUACAAAAGCAAAA  836 AS836-M1 CAGGUCUA S384- ACCUGUUUUGCUUUUGUAAUUUGAA  384 UUCAAAUUACAAAAGCAAA  837 AS837-M1 ACAGGUCU S385- CCUGUUUUGCUUUUGUAACUUGAAG  385 CUUCAAGUUACAAAAGCAA  838 AS838-M1 AACAGGUC 5386- CUGUUUUGCUUUUGUAACUUGAAGA  386 UCUUCAAGUUACAAAAGCA  839 AS839-M1 AAACAGGU S387- UGUUUUGCUUUUGUAACUUUAAGAT  387 AUCUUAAAGUUACAAAAGC  840 AS840-M1 AAAACAGG S388- GUUUUGCUUUUGUAACUUGAAGATA  388 UAUCUUCAAGUUACAAAAG  841 AS841-M1 CAAAACAG S389- UUUUGCUUUUGUAACUUGAAGAUAT  389 AUAUCUUCAAGUUACAAAA  842 AS842-M1 GCAAAACA S390- UUUGCUUUUGUAACUUGAAUAUATT  390 AAUAUAUUCAAGUUACAAA  843 AS843-M1 AGCAAAAC S391- UUGCUUUUGUAACUUGAAGAUAUTT  391 AAAUAUCUUCAAGUUACAA  844 AS844-M1 AAGCAAAA S392- UGCUUUUGUAACUUGAAGAUAUUTA  392 UAAAUAUCUUCAAGUUACA  845 AS845-M1 AAAGCAAA S393- GCUUUUGUAACUUGAAGAUAUUUAT  393 AUAAAUAUCUUCAAGUUAC  846 AS846-M1 AAAAGCAA S394- CUUUUGUAACUUGAAGAUAUUUATT  394 AAUAAAUAUCUUCAAGUUA  847 AS847-M1 CAAAAGCA S395- UUUUGUAACUUGAAGAUAUUUAUTC  395 GAAUAAAUAUCUUCAAGUU  848 AS848-M1 ACAAAAGC S396- UUUGUAACUUGAAGAUAUUUAUUCT  396 AGAAUAAAUAUCUUCAAGU  849 AS849-M1 UACAAAAG S397- UUGUAACUUGAAGAUAUUUAUUCTG  397 CAGAAUAAAUAUCUUCAAG  850 AS850-M1 UUACAAAA S398- UGUAACUUGAAGAUAUUUAUUCUGG  398 CCAGAAUAAAUAUCUUCAA  851 AS851-M1 GUUACAAA S399- GUAACUUGAAGAUAUUUAUUCUGGG  399 CCCAGAAUAAAUAUCUUCA  852 AS852-M1 AGUUACAA S400- UAACUUGAAGAUAUUUAUUUUGGGT  400 ACCCAAAAUAAAUAUCUUC  853 AS853-M1 AAGUUACA S401- ACUUGAAGAUAUUUAUUCUUGGUTT  401 AAACCAAGAAUAAAUAUCU  854 AS854-M1 UCAAGUUA S402- CUUGAAGAUAUUUAUUCUGUGUUTT  402 AAAACACAGAAUAAAUAUC  855 AS855-M1 UUCAAGUU S403- UUGAAGAUAUUUAUUCUGGUUUUTG  403 CAAAAACCAGAAUAAAUAU  856 AS856-M1 CUUCAAGU S404- GAAGAUAUUUAUUCUGGGUUUUGTA  404 UACAAAACCCAGAAUAAAU  857 AS857-M1 AUCUUCAA S405- AAGAUAUUUAUUCUGGGUUUUGUAG  405 CUACAAAACCCAGAAUAAA  858 AS858-M1 UAUCUUCA S406- AUAUUUAUUCUGGGUUUUGUAGCAT  406 AUGCUACAAAACCCAGAAU  859 AS859-M1 AAAUAUCU S407- UAUUUAUUCUGGGUUUUGUAGCATT  407 AAUGCUACAAAACCCAGAA  860 AS860-M1 UAAAUAUC S408- AUUUAUUCUGGGUUUUGUAUCAUTT  408 AAAUGAUACAAAACCCAGA  861 AS861-M1 AUAAAUAU S409- UUUAUUCUGGGUUUUGUAGUAUUTT  409 AAAAUACUACAAAACCCAG  862 AS862-M1 AAUAAAUA S410- AUUCUGGGUUUUGUAGCAUUUUUAT  410 AUAAAAAUGCUACAAAACC  863 AS863-M1 CAGAAUAA S411- UUCUGGGUUUUGUAGCAUUUUUATT  411 AAUAAAAAUGCUACAAAAC  864 AS864-M1 CCAGAAUA S412- UCUGGGUUUUGUAGCAUUUUUAUTA  412 UAAUAAAAAUGCUACAAAA  865 AS865-M1 CCCAGAAU S413- CUGGGUUUUGUAGCAUUUUUAUUAA  413 UUAAUAAAAAUGCUACAAA  866 AS866-M1 ACCCAGAA S414- UGGGUUUUGUAGCAUUUUUAUUAAT  414 AUUAAUAAAAAUGCUACAA  867 AS867-M1 AACCCAGA S415- GGGUUUUGUAGCAUUUUUAUUAATA  415 UAUUAAUAAAAAUGCUACA  868 AS868-M1 AAACCCAG S416- GGUUUUGUAGCAUUUUUAUUAAUAT  416 AUAUUAAUAAAAAUGCUAC  869 AS869-M1 AAAACCCA S417- GUUUUGUAGCAUUUUUAUUAAUATG  417 CAUAUUAAUAAAAAUGCUA  870 AS870-M1 CAAAACCC S418- UUUUGUAGCAUUUUUAUUAAUAUGG  418 CCAUAUUAAUAAAAAUGCU  871 AS871-M1 ACAAAACC S419- UUUGUAGCAUUUUUAUUAAUAUGGT  419 ACCAUAUUAAUAAAAAUGC  872 AS872-M1 UACAAAAC S420- UUGUAGCAUUUUUAUUAAUAUGGTG  420 CACCAUAUUAAUAAAAAUG  873 AS873-M1 CUACAAAA S421- UGUAGCAUUUUUAUUAAUAUGGUGA  421 UCACCAUAUUAAUAAAAAU  874 AS874-M1 GCUACAAA S422- GUAGCAUUUUUAUUAAUAUUGUGAC  422 GUCACAAUAUUAAUAAAAA  875 AS875-M1 UGCUACAA S423- UAGCAUUUUUAUUAAUAUGUUGACT  423 AGUCAACAUAUUAAUAAAA  876 AS876-M1 AUGCUACA S424- AGCAUUUUUAUUAAUAUGGUGACTT  424 AAGUCACCAUAUUAAUAAA  877 AS877-M1 AAUGCUAC S425- GCAUUUUUAUUAAUAUGGUUACUTT  425 AAAGUAACCAUAUUAAUAA  878 AS878-M1 AAAUGCUA S426- CAUUUUUAUUAAUAUGGUGACUUTT  426 AAAAGUCACCAUAUUAAUA  879 AS879-M1 AAAAUGCU S427- AUUUUUAUUAAUAUGGUGAUUUUTT  427 AAAAAAUCACCAUAUUAAU  880 AS880-M1 AAAAAUGC S428- UUUUUAUUAAUAUGGUGACUUUUTA  428 UAAAAAGUCACCAUAUUAA  881 AS881-M1 UAAAAAUG S429- UUUUAUUAAUAUGGUGACUUUUUAA  429 UUAAAAAGUCACCAUAUUA  882 AS882-M1 AUAAAAAU S430- UUUAUUAAUAUGGUGACUUUUUAAA  430 UUUAAAAAGUCACCAUAUU  883 AS883-M1 AAUAAAAA S431- UUAUUAAUAUGGUGACUUUUUAAAA  431 UUUUAAAAAGUCACCAUAU  884 AS884-M1 UAAUAAAA S432- UAUUAAUAUGGUGACUUUUUAAAAT  432 AUUUUAAAAAGUCACCAUA  885 AS885-M1 UUAAUAAA S433- AUUAAUAUGGUGACUUUUUAAAATA  433 UAUUUUAAAAAGUCACCAU  886 AS886-M1 AUUAAUAA S434- UUAAUAUGGUGACUUUUUAAAAUAA  434 UUAUUUUAAAAAGUCACCA  887 AS887-M1 UAUUAAUA S435- UAAUAUGGUGACUUUUUAAAAUAAA  435 UUUAUUUUAAAAAGUCACC  888 AS888-M1 AUAUUAAU S436- AAUAUGGUGACUUUUUAAAAUAAAA  436 UUUUAUUUUAAAAAGUCAC  889 AS889-M1 CAUAUUAA S437- AUAUGGUGACUUUUUAAAAUAAAAA  437 UUUUUAUUUUAAAAAGUCA  890 AS890-M1 CCAUAUUA S438- UAUGGUGACUUUUUAAAAUAAAAAC  438 GUUUUUAUUUUAAAAAGUC  891 AS891-M1 ACCAUAUU S439- AUGGUGACUUUUUAAAAUAAAAACA  439 UGUUUUUAUUUUAAAAAGU  892 AS892-M1 CACCAUAU S440- UGGUGACUUUUUAAAAUAAAAACAA  440 UUGUUUUUAUUUUAAAAAG  893 AS893-M1 UCACCAUA S441- GGUGACUUUUUAAAAUAAAAACAAA  441 UUUGUUUUUAUUUUAAAAA  894 AS894-M1 GUCACCAU S442- GUGACUUUUUAAAAUAAAAACAAAC  442 GUUUGUUUUUAUUUUAAAA  895 AS895-M1 AGUCACCA S443- UGACUUUUUAAAAUAAAAAUAAACA  443 UGUUUAUUUUUAUUUUAAA  896 AS896-M1 AAGUCACC S444- GACUUUUUAAAAUAAAAACAAACAA  444 UUGUUUGUUUUUAUUUUAA  897 AS897-M1 AAAGUCAC S445- ACUUUUUAAAAUAAAAACAAACAAA  445 UUUGUUUGUUUUUAUUUUA  898 AS898-M1 AAAAGUCA S446- UUUUAAAAUAAAAACAAACAAACGT  446 ACGUUUGUUUGUUUUUAUU  899 AS899-M1 UUAAAAAG S447- UUUAAAAUAAAAACAAACAAACGTT  447 AACGUUUGUUUGUUUUUAU  900 AS900-M1 UUUAAAAA S448- UUAAAAUAAAAACAAACAAACGUTG  448 CAACGUUUGUUUGUUUUUA  901 AS901-M1 UUUUAAAA S449- UAAAAUAAAAACAAACAAAUGUUGT  449 ACAACAUUUGUUUGUUUUU  902 AS902-M1 AUUUUAAA S450- AAAAACAAACAAACGUUGUUCUAAC  450 GUUAGAACAACGUUUGUUU  903 AS903-M1 GUUUUUAU S451- CAAACAAACGUUGUCCUAAUAAAAA  451 UUUUUAUUAGGACAACGUU  904 AS904-M1 UGUUUGUU S452- AAACAAACGUUGUCCUAACAAAAAA  452 UUUUUUGUUAGGACAACGU  905 AS905-M1 UUGUUUGU S453- AACAAACGUUGUCCUAACAAAAAAA  453 UUUUUUUGUUAGGACAACG  906 AS906-M1 UUUGUUUG S907- CUCCAGGCGGUCCUGGUGGUCGCTG  907 CAGCGACCACCAGGACCGC 1030 AS1030-M1 CUGGAGCU S908- UCCAGGCGGUCCUGGUGGCUGCUGC  908 GCAGCAGCCACCAGGACCG 1031 AS1031-M1 CCUGGAGC S909- GCCGCUGCCACUGCUGCUGUUGCTG  909 CAGCAACAGCAGCAGUGGC 1032 AS1032-M1 AGCGGCCA S910- CCGCUGCCACUGCUGCUGCUGCUGC  910 GCAGCAGCAGCAGCAGUGG 1033 AS1033-M1 CAGCGGCC S911- GCCCGUGCGCAGGAGGACGAGGACG  911 CGUCCUCGUCCUCCUGCGC 1034 AS1034-M1 ACGGGCGC S912- CCCGUGCGCAGGAGGACGAUGACGG  912 CCGUCAUCGUCCUCCUGCG 1035 AS1035-M1 CACGGGCG S913- CCGUGCGCAGGAGGACGAGUACGGC  913 GCCGUACUCGUCCUCCUGC 1036 AS1036-M1 GCACGGGC S914- CGUGCGCAGGAGGACGAGGACGGCG  914 CGCCGUCCUCGUCCUCCUG 1037 AS1037-M1 CGCACGGG S915- GUGCGCAGGAGGACGAGGAUGGCGA  915 UCGCCAUCCUCGUCCUCCU 1038 AS1038-M1 GCGCACGG S916- UGCGCAGGAGGACGAGGACUGCGAC  916 GUCGCAGUCCUCGUCCUCC 1039 AS1039-M1 UGCGCACG S917- GCGCAGGAGGACGAGGACGUCGACT  917 AGUCGACGUCCUCGUCCUC 1040 AS1040-M1 CUGCGCAC S918- GGAGGACGAGGACGGCGACUACGAG  918 CUCGUAGUCGCCGUCCUCG 1041 AS1041-M1 UCCUCCUG S919- GCGUUCCGAGGAGGACGGCUUGGCC  919 GGCCAAGCCGUCCUCCUCG 1042 AS1042-M1 GAACGCAA S920- CGUUCCGAGGAGGACGGCCUGGCCG  920 CGGCCAGGCCGUCCUCCUC 1043 AS1043-M1 GGAACGCA S921- GUUCCGAGGAGGACGGCCUUGCCGA  921 UCGGCAAGGCCGUCCUCCU 1044 AS1044-M1 CGGAACGC S922- UUCCGAGGAGGACGGCCUGUCCGAA  922 UUCGGACAGGCCGUCCUCC 1045 AS1045-M1 UCGGAACG S923- UCCGAGGAGGACGGCCUGGUCGAAG  923 CUUCGACCAGGCCGUCCUC 1046 AS1046-M1 CUCGGAAC S924- CCGAGGAGGACGGCCUGGCUGAAGC  924 GCUUCAGCCAGGCCGUCCU 1047 AS1047-M1 CCUCGGAA S925- CGAGGAGGACGGCCUGGCCUAAGCA  925 UGCUUAGGCCAGGCCGUCC 1048 AS1048-M1 UCCUCGGA S926- GAGGAGGACGGCCUGGCCGAAGCAC  926 GUGCUUCGGCCAGGCCGUC 1049 AS1049-M1 CUCCUCGG S927- GCCACCUUCCACCGCUGCGUCAAGG  927 CCUUGACGCAGCGGUGGAA 1050 AS1050-M1 GGUGGCUG S928- CCACCUUCCACCGCUGCGCUAAGGA  928 UCCUUAGCGCAGCGGUGGA 1051 AS1051-M1 AGGUGGCU S929- CACCUUCCACCGCUGCGCCAAGGAT  929 AUCCUUGGCGCAGCGGUGG 1052 AS1052-M1 AAGGUGGC S930- ACCUUCCACCGCUGCGCCAAGGATC  930 GAUCCUUGGCGCAGCGGUG 1053 AS1053-M1 GAAGGUGG S931- AGCGCACUGCCCGCCGCCUUCAGGC  931 GCCUGAAGGCGGCGGGCAG 1054 AS1054-M1 UGCGCUCU S932- GCGCACUGCCCGCCGCCUGUAGGCC  932 GGCCUACAGGCGGCGGGCA 1055 AS1055-M1 GUGCGCUC S933- CGCACUGCCCGCCGCCUGCAGGCCC  933 GGGCCUGCAGGCGGCGGGC 1056 AS1056-M1 AGUGCGCU S934- GCACUGCCCGCCGCCUGCAUGCCCA  934 UGGGCAUGCAGGCGGCGGG 1057 AS1057-M1 CAGUGCGC S935- CACUGCCCGCCGCCUGCAGUCCCAG  935 CUGGGACUGCAGGCGGCGG 1058 AS1058-M1 GCAGUGCG S936- ACUGCCCGCCGCCUGCAGGUCCAGG  936 CCUGGACCUGCAGGCGGCG 1059 AS1059-M1 GGCAGUGC S937- CUGCCCGCCGCCUGCAGGCUCAGGC  937 GCCUGAGCCUGCAGGCGGC 1060 AS1060-M1 GGGCAGUG S938- UGCCCGCCGCCUGCAGGCCUAGGCT  938 AGCCUAGGCCUGCAGGCGG 1061 AS1061-M1 CGGGCAGU S939- GCCCGCCGCCUGCAGGCCCAGGCTG  939 CAGCCUGGGCCUGCAGGCG 1062 AS1062-M1 GCGGGCAG S940- CCCGCCGCCUGCAGGCCCAUGCUGC  940 GCAGCAUGGGCCUGCAGGC 1063 AS1063-M1 GGCGGGCA S941- UGGCGACCUGCUGGAGCUGUCCUTG  941 CAAGGACAGCUCCAGCAGG 1064 AS1064-M1 UCGCCACU S942- GGCGACCUGCUGGAGCUGGUCUUGA  942 UCAAGACCAGCUCCAGCAG 1065 AS1065-M1 GUCGCCAC S943- GCGACCUGCUGGAGCUGGCUUUGAA  943 UUCAAAGCCAGCUCCAGCA 1066 AS1066-M1 GGUCGCCA S944- CGACCUGCUGGAGCUGGCCUUGAAG  944 CUUCAAGGCCAGCUCCAGC 1067 AS1067-M1 AGGUCGCC S945- GAGGCAGCCUGGUGGAGGUUUAUC  945 AGAUAAACCUCCACCAGGC 1068 AS1068-M1 UGCCUCCG S946- AGGCAGCCUGGUGGAGGUGUAUCTC  946 GAGAUACACCUCCACCAGG 1069 AS1069-M1 CUGCCUCC S947- UGUGCCCGAGGAGGACGGGACCCGC  947 GCGGGUCCCGUCCUCCUCG 1070 AS1070-M1 GGCACAUU S948- GUGCCCGAGGAGGACGGGAUCCGCT  948 AGCGGAUCCCGUCCUCCUC 1071 AS1071-M1 GGGCACAU S949- UGCCCGAGGAGGACGGGACUCGCTT  949 AAGCGAGUCCCGUCCUCCU 1072 AS1072-M1 CGGGCACA S950- GCCCGAGGAGGACGGGACCUGCUTC  950 GAAGCAGGUCCCGUCCUCC 1073 AS1073-M1 UCGGGCAC S951- CCCGAGGAGGACGGGACCCUCUUCC  951 GGAAGAGGGUCCCGUCCUC 1074 AS1074-M1 CUCGGGCA S952- CCGAGGAGGACGGGACCCGUUUCCA  952 UGGAAACGGGUCCCGUCCU 1075 AS1075-M1 CCUCGGGC S953- CGAGGAGGACGGGACCCGCUUCCAC  953 GUGGAAGCGGGUCCCGUCC 1076 AS1076-M1 UCCUCGGG S954- GGCAGGGGUGGUCAGCGGCUGGGAT  954 AUCCCAGCCGCUGACCACC 1077 AS1077-M1 CCUGCCAG S955- GCAGGGGUGGUCAGCGGCCUGGATG  955 CAUCCAGGCCGCUGACCAC 1078 AS1078-M1 CCCUGCCA S956- CAGGGGUGGUCAGCGGCCGUGAUGC  956 GCAUCACGGCCGCUGACCA 1079 AS1079-M1 CCCCUGCC S957- GUGCUGCUGCCCCUGGCGGUUGGGT  957 ACCCAACCGCCAGGGGCAG 1080 AS1080-M1 CAGCACCA S958- UGCUGCUGCCCCUGGCGGGUGGGTA  958 UACCCACCCGCCAGGGGCA 1081 AS1081-M1 GCAGCACC S959- GCUGCUGCCCCUGGCGGGUUGGUAC  959 GUACCAACCCGCCAGGGGC 1082 AS1082-M1 AGCAGCAC S960- CUGCUGCCCCUGGCGGGUGUGUACA  960 UGUACACACCCGCCAGGGG 1083 AS1083-M1 CAGCAGCA S961- UGCUGCCCCUGGCGGGUGGUUACAG  961 CUGUAACCACCCGCCAGGG 1084 AS1084-M1 GCAGCAGC S962- GCUGCCCCUGGCGGGUGGGUACAGC  962 GCUGUACCCACCCGCCAGG 1085 AS1085-M1 GGCAGCAG S963- CUGCCCCUGGCGGGUGGGUACAGCC  963 GGCUGUACCCACCCGCCAG 1086 AS1086-M1 GGGCAGCA S964- UGCCCCUGGCGGGUGGGUAUAGCCG  964 CGGCUAUACCCACCCGCCA 1087 AS1087-M1 GGGGCAGC S965- GCCCCUGGCGGGUGGGUACAGCCGC  965 GCGGCUGUACCCACCCGCC 1088 AS1088-M1 AGGGGCAG S966- UCAACGCCGCCUGCCAGCGUCUGGC  966 GCCAGACGCUGGCAGGCGG 1089 AS1089-M1 CGUUGAGG S967- CAACGCCGCCUGCCAGCGCUUGGCG  967 CGCCAAGCGCUGGCAGGCG 1090 AS1090-M1 GCGUUGAG S968- AACGCCGCCUGCCAGCGCCUGGCGA  968 UCGCCAGGCGCUGGCAGGC 1091 AS1091-M1 GGCGUUGA S969- ACGCCGCCUGCCAGCGCCUUGCGAG  969 CUCGCAAGGCGCUGGCAGG 1092 AS1092-M1 CGGCGUUG S970- CGCCGCCUGCCAGCGCCUGUCGAGG  970 CCUCGACAGGCGCUGGCAG 1093 AS1093-M1 GCGGCGUU S971- GCCGCCUGCCAGCGCCUGGUGAGGG  971 CCCUCACCAGGCGCUGGCA 1094 AS1094-M1 GGCGGCGU S972- CCGCCUGCCAGCGCCUGGCUAGGGC  972 GCCCUAGCCAGGCGCUGGC 1095 AS1095-M1 AGGCGGCG S973- CGCCUGCCAGCGCCUGGCGAGGGCT  973 AGCCCUCGCCAGGCGCUGG 1096 AS1096-M1 CAGGCGGC S974- GCCUGCCAGCGCCUGGCGAUGGCTG  974 CAGCCAUCGCCAGGCGCUG 1097 AS1097-M1 GCAGGCGG S975- CCAGCGCCUGGCGAGGGCUUGGGTC  975 GACCCAAGCCCUCGCCAGG 1098 AS1098-M1 CGCUGGCA S976- CAGCGCCUGGCGAGGGCUGUGGUCG  976 CGACCACAGCCCUCGCCAG 1099 AS1099-M1 GCGCUGGC S977- AGCGCCUGGCGAGGGCUGGUGUCGT  977 ACGACACCAGCCCUCGCCA 1100 AS1100-M1 GGCGCUGG S978- GCGCCUGGCGAGGGCUGGGUUCGTG  978 CACGAACCCAGCCCUCGCC 1101 AS1101-M1 AGGCGCUG S979- CGCCUGGCGAGGGCUGGGGUCGUGC  979 GCACGACCCCAGCCCUCGC 1102 AS1102-M1 CAGGCGCU S980- GCGAGGGCUGGGGUCGUGCUGGUCA  980 UGACCAGCACGACCCCAGC 1103 AS1103-M1 CCUCGCCA S981- AUGCCUGCCUCUACUCCCCAGCCTC  981 GAGGCUGGGGAGUAGAGGC 1104 AS1104-M1 AGGCAUCG S982- GCCUCUACUCCCCAGCCUCAGCUCC  982 GGAGCUGAGGCUGGGGAGU 1105 AS1105-M1 AGAGGCAG S983- GACCUCUUUGCCCCAGGGGAGGACA  983 UGUCCUCCCCUGGGGCAAA 1106 AS1106-M1 GAGGUCCA S984- CUUUGCCCCAGGGGAGGACAUCATT  984 AAUGAUGUCCUCCCCUGGG 1107 AS1107-M1 GCAAAGAG S985- UUUGCCCCAGGGGAGGACAUCAUTG  985 CAAUGAUGUCCUCCCCUGG 1108 AS1108-M1 GGCAAAGA S986- UUGCCCCAGGGGAGGACAUUAUUGG  986 CCAAUAAUGUCCUCCCCUG 1109 AS1109-M1 GGGCAAAG S987- UGCCCCAGGGGAGGACAUCAUUGGT  987 ACCAAUGAUGUCCUCCCCU 1110 AS1110-M1 GGGGCAAA S988- GCCCCAGGGGAGGACAUCAUUGGTG  988 CACCAAUGAUGUCCUCCCC 1111 AS1111-M1 UGGGGCAA S989- ACACGGAUGGCCACAGCCGUCGCCC  989 GGGCGACGGCUGUGGCCAU 1112 AS1112-M1 CCGUGUAG S990- CUCCAGGAGUGGGAAGCGGUGGGGC  990 GCCCCACCGCUUCCCACUC 1113 AS1113-M1 CUGGAGAA S991- UCCAGGAGUGGGAAGCGGCUGGGCG  991 CGCCCAGCCGCUUCCCACU 1114 AS1114-M1 CCUGGAGA S992- CCAGGAGUGGGAAGCGGCGUGGCGA  992 UCGCCACGCCGCUUCCCAC 1115 AS1115-M1 UCCUGGAG S993- CAGGAGUGGGAAGCGGCGGUGCGAG  993 CUCGCACCGCCGCUUCCCA 1116 AS1116-M1 CUCCUGGA S994- AGGAGUGGGAAGCGGCGGGUCGAGC  994 GCUCGACCCGCCGCUUCCC 1117 AS1117-M1 ACUCCUGG S995- GGAGUGGGAAGCGGCGGGGUGAGCG  995 CGCUCACCCCGCCGCUUCC 1118 AS1118-M1 CACUCCUG S996- GAGUGGGAAGCGGCGGGGCUAGCGC  996 GCGCUAGCCCCGCCGCUUC 1119 AS1119-M1 CCACUCCU S997- AGUGGGAAGCGGCGGGGCGAGCGCA  997 UGCGCUCGCCCCGCCGCUU 1120 AS1120-M1 CCCACUCC S998- GAAGCGGCGGGGCGAGCGCAUGGAG  998 CUCCAUGCGCUCGCCCCGC 1121 AS1121-M1 CGCUUCCC S999- AAGCGGCGGGGCGAGCGCAUGGAGG  999 CCUCCAUGCGCUCGCCCCG 1122 AS1122-M1 CCGCUUCC S1000- AGCGGCGGGGCGAGCGCAUUGAGGC 1000 GCCUCAAUGCGCUCGCCCC 1123 AS1123-M1 GCCGCUUC S1001- GGUGCUGCCUGCUACCCCAUGCCAA 1001 UUGGCAUGGGGUAGCAGGC 1124 AS1124-M1 AGCACCUG S1002- GUGCUGCCUGCUACCCCAGUCCAAC 1002 GUUGGACUGGGGUAGCAGG 1125 AS1125-M1 CAGCACCU S1003- UGCUGCCUGCUACCCCAGGUCAACT 1003 AGUUGACCUGGGGUAGCAG 1126 AS1126-M1 GCAGCACC S1004- GGGCCACGUCCUCACAGGCUGCAGC 1004 GCUGCAGCCUGUGAGGACG 1127 AS1127-M1 UGGCCCUG S1005- GGCCACGUCCUCACAGGCUUCAGCT 1005 AGCUGAAGCCUGUGAGGAC 1128 AS1128-M1 GUGGCCCU S1006- GCCACGUCCUCACAGGCUGUAGCTC 1006 GAGCUACAGCCUGUGAGGA 1129 AS1129-M1 CGUGGCCC S1007- GGCUGCAGCUCCCACUGGGAGGUGG 1007 CCACCUCCCAGUGGGAGCU 1130 AS1130-M1 GCAGCCUG S1008- GCUGCAGCUCCCACUGGGAUGUGGA 1008 UCCACAUCCCAGUGGGAGC 1131 AS1131-M1 UGCAGCCU S1009- CUGCAGCUCCCACUGGGAGUUGGAG 1009 CUCCAACUCCCAGUGGGAG 1132 AS1132-M1 CUGCAGCC S1010- UGCAGCUCCCACUGGGAGGUGGAGG 1010 CCUCCACCUCCCAGUGGGA 1133 AS1133-M1 GCUGCAGC S1011- GCAGCUCCCACUGGGAGGUUGAGGA 1011 UCCUCAACCUCCCAGUGGG 1134 AS1134-M1 AGCUGCAG S1012- CAGCUCCCACUGGGAGGUGUAGGAC 1012 GUCCUACACCUCCCAGUGG 1135 AS1135-M1 GAGCUGCA S1013- AGCUCCCACUGGGAGGUGGAGGACC 1013 GGUCCUCCACCUCCCAGUG 1136 AS1136-M1 GGAGCUGC S1014- GCUCCCACUGGGAGGUGGAUGACCT 1014 AGGUCAUCCACCUCCCAGU 1137 AS1137-M1 GGGAGCUG S1015- CUCCCACUGGGAGGUGGAGUACCTT 1015 AAGGUACUCCACCUCCCAG 1138 AS1138-M1 UGGGAGCU S1016- UCCCACUGGGAGGUGGAGGACCUTG 1016 CAAGGUCCUCCACCUCCCA 1139 AS1139-M1 GUGGGAGC S1017- UGGCACCCACAAGCCGCCUUUGCTG 1017 CAGCAAAGGCGGCUUGUGG 1140 AS1140-M1 GUGCCAAG S1018- GGCACCCACAAGCCGCCUGUGCUGA 1018 UCAGCACAGGCGGCUUGUG 1141 AS1141-M1 GGUGCCAA S1019- AGCCGCCUGUGCUGAGGCCACGAGG 1019 CCUCGUGGCCUCAGCACAG 1142 AS1142-M1 GCGGCUUG S1020- GCCGCCUGUGCUGAGGCCAUGAGGT 1020 ACCUCAUGGCCUCAGCACA 1143 AS1143-M1 GGCGGCUU S1021- CCGCCUGUGCUGAGGCCACUAGGTC 1021 GACCUAGUGGCCUCAGCAC 1144 AS1144-M1 AGGCGGCU S1022- GGGCCACAGGGAGGCCAGCAUCCAC 1022 GUGGAUGCUGGCCUCCCUG 1145 AS1145-M1 UGGCCCAC S1023- GGCCACAGGGAGGCCAGCAUCCACG 1023 CGUGGAUGCUGGCCUCCCU 1146 AS1146-M1 GUGGCCCA S1024- GCCACAGGGAGGCCAGCAUUCACGC 1024 GCGUGAAUGCUGGCCUCCC 1147 AS1147-M1 UGUGGCCC S1025- CGGCCCCUCAGGAGCAGGUUACCGT 1025 ACGGUAACCUGCUCCUGAG 1148 AS1148-M1 GGGCCGGG S1026- UGCUGCCGGAGCCGGCACCUGGCGC 1026 GCGCCAGGUGCCGGCUCCG 1149 AS1149-M1 GCAGCAGA S1027- UCACAGGCUGCUGCCCACGUGGCTG 1027 CAGCCACGUGGGCAGCAGC 1150 AS1150-M1 CUGUGAUG S1028- CACAGGCUGCUGCCCACGUUGCUGG 1028 CCAGCAACGUGGGCAGCAG 1151 AS1151-M1 CCUGUGAU S1029- GCUUCCUGCUGCCAUGCCCUAGGTC 1029 GACCUAGGGCAUGGCAGCA 1152 AS1152-M1 GGAAGCGU S1153- AACUUCAGCUCCUGCACAGUGCAGC 1153 ACUGUGCAGGAGCUGAAGU 1193 AS1193-M2 CGAAAGGCUGC UCA S1154- UGGCCCUCAUGGGCACCGUUGCAGC 1154 AACGGUGCCCAUGAGGGCC 1194 AS1194-M2 CGAAAGGCUGC AGG S1155- AGGAGGAGACCCACCUCUCUGCAGC 1155 AGAGAGGUGGGUCUCCUCC 1195 AS1195-M2 CGAAAGGCUGC UUC S1156- UGCUGGAGCUGGCCUUGAAUGCAGC 1156 AUUCAAGGCCAGCUCCAGC 1196 AS1196-M2 CGAAAGGCUGC AGG S1157- UCUGUCUUUGCCCAGAGCAUGCAGC 1157 AUGCUCUGGGCAAAGACAG 1197 AS1197-M2 CGAAAGGCUGC AGG S1158- CUGUCUUUGCCCAGAGCAUUGCAGC 1158 AAUGCUCUGGGCAAAGACA 1198 AS1198-M2 CGAAAGGCUGC GAG S1159- CUUGCCUGGAACUCACUCAUGCAGC 1159 AUGAGUGAGUUCCAGGCAA 1199 AS1199-M2 CGAAAGGCUGC GGA S1160- UUGCCUGGAACUCACUCACUGCAGC 1160 AGUGAGUGAGUUCCAGGCA 1200 AS1200-M2 CGAAAGGCUGC AGG S1161- AGAAUGACUUUUAUUGAGCUGCAGC 1161 AGCUCAAUAAAAGUCAUUC 1201 AS1201-M2 CGAAAGGCUGC UGC S1162- GAAUGACUUUUAUUGAGCUUGCAGC 1162 AAGCUCAAUAAAAGUCAUU 1202 AS1202-M2 CGAAAGGCUGC CUG S1163- AUGACUUUUAUUGAGCUCUUGCAGC 1163 AAGAGCUCAAUAAAAGUCA 1203 AS1203-M2 CGAAAGGCUGC UUC S1164- UGACUUUUAUUGAGCUCUUUGCAGC 1164 AAAGAGCUCAAUAAAAGUC 1204 AS1204-M2 CGAAAGGCUGC AUU S1165- CUUGUUCCGUGCCAGGCAUUGCAGC 1165 AAUGCCUGGCACGGAACAA 1205 AS1205-M2 CGAAAGGCUGC GAG S1166- UGUGAAAGGUGCUGAUGGCUGCAGC 1166 AGCCAUCAGCACCUUUCAC 1206 AS1206-M2 CGAAAGGCUGC ACU S1167- AUGGAGGCUUAGCUUUCUGUGCAGC 1167 ACAGAAAGCUAAGCCUCCA 1207 AS1207-M2 CGAAAGGCUGC UUA S1168- GAGGCUUAGCUUUCUGGAUUGCAGC 1168 AAUCCAGAAAGCUAAGCCU 1208 AS1208-M2 CGAAAGGCUGC CCA S1169- AGGCUUAGCUUUCUGGAUGUGCAGC 1169 ACAUCCAGAAAGCUAAGCC 1209 AS1209-M2 CGAAAGGCUGC UCC S1170- GCUUAGCUUUCUGGAUGGCAGCAGC 1170 UGCCAUCCAGAAAGCUAAG 1210 AS1210-M2 CGAAAGGCUGC CCU S1171- CCAGGCUGUGCUAGCAACAUGCAGC 1171 AUGUUGCUAGCACAGCCUG 1211 AS1211-M2 CGAAAGGCUGC GCA S1172- UGCGGGGAGCCAUCACCUAUGCAGC 1172 AUAGGUGAUGGCUCCCCGC 1212 AS1212-M2 CGAAAGGCUGC AGG S1173- CGGCAGUGUGCAGUGGUGCAGCAGC 1173 UGCACCACUGCACACUGCC 1213 AS1213-M2 CGAAAGGCUGC GAG S1174- ACAGAGGAAGAAACCUGGAAGCAGC 1174 UUCCAGGUUUCUUCCUCUG 1214 AS1214-M2 CGAAAGGCUGC UGA S1175- CAGAGGAAGAAACCUGGAAUGCAGC 1175 AUUCCAGGUUUCUUCCUCU 1215 AS1215-M2 CGAAAGGCUGC GUG S1176- AGAGGAAGAAACCUGGAACUGCAGC 1176 AGUUCCAGGUUUCUUCCUC 1216 AS1216-M2 CGAAAGGCUGC UGU S1177- UGGCGGAGAUGCUUCUAAGUGCAGC 1177 ACUUAGAAGCAUCUCCGCC 1217 AS1217-M2 CGAAAGGCUGC AGG S1178- UUACAGCCAACUUUUCUAGAGCAGC 1178 UCUAGAAAAGUUGGCUGUA 1218 AS1218-M2 CGAAAGGCUGC AAA S1179- CUGUUUUGCUUUUGUAACUUGCAGC 1179 AAGUUACAAAAGCAAAACA 1219 AS1219-M2 CGAAAGGCUGC GGU S1180- UGUUUUGCUUUUGUAACUUUGCAGC 1180 AAAGUUACAAAAGCAAAAC 1220 AS1220-M2 CGAAAGGCUGC AGG S1181- UUUGCUUUUGUAACUUGAAUGCAGC 1181 AUUCAAGUUACAAAAGCAA 1221 AS1221-M2 CGAAAGGCUGC AAC S1182- UUUGUAGCAUUUUUAUUAAUGCAGC 1182 AUUAAUAAAAAUGCUACAA 1222 AS1222-M2 CGAAAGGCUGC AAC S1183- UGUAGCAUUUUUAUUAAUAUGCAGC 1183 AUAUUAAUAAAAAUGCUAC 1223 AS1223-M2 CGAAAGGCUGC AAA S1184- GUAGCAUUUUUAUUAAUAUUGCAGC 1184 AAUAUUAAUAAAAAUGCUA 1224 AS1224-M2 CGAAAGGCUGC CAA S1185- AUUAAUAUGGUGACUUUUUAGCAGC 1185 UAAAAAGUCACCAUAUUAA 1225 AS1225-M2 CGAAAGGCUGC UAA S1186- UUAAUAUGGUGACUUUUUAAGCAGC 1186 UUAAAAAGUCACCAUAUUA 1226 AS1226-M2 CGAAAGGCUGC AUA S1187- AAUAUGGUGACUUUUUAAAAGCAGC 1187 UUUUAAAAAGUCACCAUAU 1227 AS1227-M2 CGAAAGGCUGC UAA S1188- AUAUGGUGACUUUUUAAAAUGCAGC 1188 AUUUUAAAAAGUCACCAUA 1228 AS1228-M2 CGAAAGGCUGC UUA S1189- UAUGGUGACUUUUUAAAAUAGCAGC 1189 UAUUUUAAAAAGUCACCAU 1229 AS1229-M2 CGAAAGGCUGC AUU S1190- AUGGUGACUUUUUAAAAUAAGCAGC 1190 UUAUUUUAAAAAGUCACCA 1230 AS1230-M2 CGAAAGGCUGC UAU S1191- UGGUGACUUUUUAAAAUAAAGCAGC 1191 UUUAUUUUAAAAAGUCACC 1231 AS1231-M2 CGAAAGGCUGC AUA S1192- GUGACUUUUUAAAAUAAAAAGCAGC 1192 UUUUUAUUUUAAAAAGUCA 1232 AS1232-M2 CGAAAGGCUGC CCA S1153- AACUUCAGCUCCUGCACAGUGCAGC 1153 ACUGUGCAGGAGCUGAAGU 1193 AS1193-M3 CGAAAGGCUGC UCA S1154- UGGCCCUCAUGGGCACCGUUGCAGC 1154 AACGGUGCCCAUGAGGGCC 1194 AS1194-M3 CGAAAGGCUGC AGG S1155- AGGAGGAGACCCACCUCUCUGCAGC 1155 AGAGAGGUGGGUCUCCUCC 1195 AS1195-M3 CGAAAGGCUGC UUC S1156- UGCUGGAGCUGGCCUUGAAUGCAGC 1156 AUUCAAGGCCAGCUCCAGC 1196 AS1196-M3 CGAAAGGCUGC AGG S1157- UCUGUCUUUGCCCAGAGCAUGCAGC 1157 AUGCUCUGGGCAAAGACAG 1197 AS1197-M3 CGAAAGGCUGC AGG S1158- CUGUCUUUGCCCAGAGCAUUGCAGC 1158 AAUGCUCUGGGCAAAGACA 1198 AS1198-M3 CGAAAGGCUGC GAG S1159- CUUGCCUGGAACUCACUCAUGCAGC 1159 AUGAGUGAGUUCCAGGCAA 1199 AS1199-M3 CGAAAGGCUGC GGA S1160- UUGCCUGGAACUCACUCACUGCAGC 1160 AGUGAGUGAGUUCCAGGCA 1200 AS1200-M3 CGAAAGGCUGC AGG S1161- AGAAUGACUUUUAUUGAGCUGCAGC 1161 AGCUCAAUAAAAGUCAUUC 1201 AS1201-M3 CGAAAGGCUGC UGC S1162- GAAUGACUUUUAUUGAGCUUGCAGC 1162 AAGCUCAAUAAAAGUCAUU 1202 AS1202-M3 CGAAAGGCUGC CUG S1163- AUGACUUUUAUUGAGCUCUUGCAGC 1163 AAGAGCUCAAUAAAAGUCA 1203 AS1203-M3 CGAAAGGCUGC UUC S1164- UGACUUUUAUUGAGCUCUUUGCAGC 1164 AAAGAGCUCAAUAAAAGUC 1204 AS1204-M3 CGAAAGGCUGC AUU S1165- CUUGUUCCGUGCCAGGCAUUGCAGC 1165 AAUGCCUGGCACGGAACAA 1205 AS1205-M3 CGAAAGGCUGC GAG S1166- UGUGAAAGGUGCUGAUGGCUGCAGC 1166 AGCCAUCAGCACCUUUCAC 1206 AS1206-M3 CGAAAGGCUGC CAU S1167- AUGGAGGCUUAGCUUUCUGUGCAGC 1167 ACAGAAAGCUAAGCCUCCA 1207 AS1207-M3 CGAAAGGCUGC UUA S1168- GAGGCUUAGCUUUCUGGAUUGCAGC 1168 AAUCCAGAAAGCUAAGCCU 1208 AS1208-M3 CGAAAGGCUGC CCA S1169- AGGCUUAGCUUUCUGGAUGUGCAGC 1169 ACAUCCAGAAAGCUAAGCC 1209 AS1209-M3 CGAAAGGCUGC UCC S1170- GCUUAGCUUUCUGGAUGGCAGCAGC 1170 UGCCAUCCAGAAAGCUAAG 1210 AS1210-M3 CGAAAGGCUGC CCU S1171- CCAGGCUGUGCUAGCAACAUGCAGC 1171 AUGUUGCUAGCACAGCCUG 1211 AS1211-M3 CGAAAGGCUGC GCA S1172- UGCGGGGAGCCAUCACCUAUGCAGC 1172 AUAGGUGAUGGCUCCCCGC 1212 AS1212-M3 CGAAAGGCUGC AGG S1173- CGGCAGUGUGCAGUGGUGCAGCAGC 1173 UGCACCACUGCACACUGCC 1213 AS1213-M3 CGAAAGGCUGC GAG S1174- ACAGAGGAAGAAACCUGGAAGCAGC 1174 UUCCAGGUUUCUUCCUCUG 1214 AS1214-M3 CGAAAGGCUGC UGA S1175- CAGAGGAAGAAACCUGGAAUGCAGC 1175 AUUCCAGGUUUCUUCCUCU 1215 AS1215-M3 CGAAAGGCUGC GUG S1176- AGAGGAAGAAACCUGGAACUGCAGC 1176 AGUUCCAGGUUUCUUCCUC 1216 AS1216-M3 CGAAAGGCUGC UGU S1177- UGGCGGAGAUGCUUCUAAGUGCAGC 1177 ACUUAGAAGCAUCUCCGCC 1217 AS1217-M3 CGAAAGGCUGC AGG S1178- UUACAGCCAACUUUUCUAGAGCAGC 1178 UCUAGAAAAGUUGGCUGUA 1218 AS1218-M3 CGAAAGGCUGC AAA S1179- CUGUUUUGCUUUUGUAACUUGCAGC 1179 AAGUUACAAAAGCAAAACA 1219 AS1219-M3 CGAAAGGCUGC GGU S1180- UGUUUUGCUUUUGUAACUUUGCAGC 1180 AAAGUUACAAAAGCAAAAC 1220 AS1220-M3 CGAAAGGCUGC AGG S1181- UUUGCUUUUGUAACUUGAAUGCAGC 1181 AUUCAAGUUACAAAAGCAA 1221 AS1221-M3 CGAAAGGCUGC AAC S1182- UUUGUAGCAUUUUUAUUAAUGCAGC 1182 AUUAAUAAAAAUGCUACAA 1222 AS1222-M3 CGAAAGGCUGC AAC S1183- UGUAGCAUUUUUAUUAAUAUGCAGC 1183 AUAUUAAUAAAAAUGCUAC 1223 AS1223-M3 CGAAAGGCUGC AAA S1184- GUAGCAUUUUUAUUAAUAUUGCAGC 1184 AAUAUUAAUAAAAAUGCUA 1224 AS1224-M3 CGAAAGGCUGC CAA S1185- AUUAAUAUGGUGACUUUUUAGCAGC 1185 UAAAAAGUCACCAUAUUAA 1225 AS1225-M3 CGAAAGGCUGC UAA S1186- UUAAUAUGGUGACUUUUUAAGCAGC 1186 UUAAAAAGUCACCAUAUUA 1226 AS1226-M3 CGAAAGGCUGC AUA S1187- AAUAUGGUGACUUUUUAAAAGCAGC 1187 UUUUAAAAAGUCACCAUAU 1227 AS1227-M3 CGAAAGGCUGC UAA S1188- AUAUGGUGACUUUUUAAAAUGCAGC 1188 AUUUUAAAAAGUCACCAUA 1228 AS1228-M3 CGAAAGGCUGC UUA S1189- UAUGGUGACUUUUUAAAAUAGCAGC 1189 UAUUUUAAAAAGUCACCAU 1229 AS1229-M3 CGAAAGGCUGC AUU S1190- AUGGUGACUUUUUAAAAUAAGCAGC 1190 UUAUUUUAAAAAGUCACCA 1230 AS1230-M3 CGAAAGGCUGC UAU S1191- UGGUGACUUUUUAAAAUAAAGCAGC 1191 UUUAUUUUAAAAAGUCACC 1231 AS1231-M3 CGAAAGGCUGC AUA S1192- GUGACUUUUUAAAAUAAAAAGCAGC 1192 UUUUUAUUUUAAAAAGUCA 1232 AS1232-M3 CGAAAGGCUGC CCA S1180- UGUUUUGCUUUUGUAACUUUGCAGC 1180 AAAGUUACAAAAGCAAAAC 1220 AS1220-M4 CGAAAGGCUGC AGG S1163- AUGACUUUUAUUGAGCUCUUGCAGC 1163 AAGAGCUCAAUAAAAGUCA 1203 AS1203-M4 CGAAAGGCUGC UUC S1181- UUUGCUUUUGUAACUUGAAUGCAGC 1181 AUUCAAGUUACAAAAGCAA 1221 AS1221-M4 CGAAAGGCUGC AAC S1248- GCUGGGCUCCUCAUUUUUAUGCAGC 1248 AUAAAAAUGAGGAGCCCAG 1257 AS1257-M4 CGAAAGGCUGC CGG S1249- GCUGGCGGAGAUGCUUCUAAGCAGC 1249 UUAGAAGCAUCUCCGCCAG 1258 AS1258-M4 CGAAAGGCUGC CGG S1250- UUUACAGCCAACUUUUCUAUGCAGC 1250 AUAGAAAAGUUGGCUGUAA 1259 AS1259-M4 CGAAAGGCUGC AGG S1251- GGCUGGGCUCCUCAUUUUUAGCAGC 1251 UAAAAAUGAGGAGCCCAGC 1260 AS1260-M4 CGAAAGGCUGC CGG S1252- AGCACGGAACCACAGCCACUGCAGC 1252 AGUGGCUGUGGUUCCGUGC 1261 AS1261-M4 CGAAAGGCUGC UGG S1253- AAUGACUUUUAUUGAGCUCUGCAGC 1253 AGAGCUCAAUAAAAGUCAU 1262 AS1262-M4 CGAAAGGCUGC UGG S1254- UUUUGUAGCAUUUUUAUUAAGCAGC 1254 UUAAUAAAAAUGCUACAAA 1263 AS1263-M4 CGAAAGGCUGC AGG S1255- GCUUGCCUGGAACUCACUCAGCAGC 1255 UGAGUGAGUUCCAGGCAAG 1264 AS1264-M4 CGAAAGGCUGC CGG S1256- UGGAGGCUUAGCUUUCUGGAGCAGC 1256 UCCAGAAAGCUAAGCCUCC 1265 AS1265-M4 CGAAAGGCUGC AGG S1180- UGUUUUGCUUUUGUAACUUUGCAGC 1180 AAAGUUACAAAAGCAAAAC 1220 AS1220-M4 CGAAAGGCUGC AGG S1180- UGUUUUGCUUUUGUAACUUUGCAGC 1180 AAAGUUACAAAAGCAAAAC 1220 AS1220-M5 CGAAAGGCUGC AGG S1164- UGACUUUUAUUGAGCUCUUUGCAGC 1164 AAAGAGCUCAAUAAAAGUC 1204 AS1204-M5 CGAAAGGCUGC AUU S1178- UUACAGCCAACUUUUCUAGAGCAGC 1178 UCUAGAAAAGUUGGCUGUA 1218 AS1218-M6 CGAAAGGCUGC AAA S1178- UUACAGCCAACUUUUCUAGAGCAGC 1178 UCUAGAAAAGUUGGCUGUA 1218 AS1218-M5 CGAAAGGCUGC AAA S1179- CUGUUUUGCUUUUGUAACUUGCAGC 1179 AAGUUACAAAAGCAAAACA 1219 AS1219-M6 CGAAAGGCUGC GGU S1179- CUGUUUUGCUUUUGUAACUUGCAGC 1179 AAGUUACAAAAGCAAAACA 1219 AS1219-M5 CGAAAGGCUGC GGU S1181- UUUGCUUUUGUAACUUGAAUGCAGC 1181 AUUCAAGUUACAAAAGCAA 1221 AS1221-M5 CGAAAGGCUGC AAC S1182- UUUGUAGCAUUUUUAUUAAUGCAGC 1182 AUUAAUAAAAAUGCUACAA 1222 AS1222-M5 CGAAAGGCUGC AAC S1183- UGUAGCAUUUUUAUUAAUAUGCAGC 1183 AUAUUAAUAAAAAUGCUAC 1223 AS1223-M5 CGAAAGGCUGC AAA S1187- AAUAUGGUGACUUUUUAAAAGCAGC 1187 UUUUAAAAAGUCACCAUAU 1227 AS1227-M5 CGAAAGGCUGC UAA S1188- AUAUGGUGACUUUUUAAAAUGCAGC 1188 AUUUUAAAAAGUCACCAUA 1228 AS1228-M5 CGAAAGGCUGC UUA S1189- UAUGGUGACUUUUUAAAAUAGCAGC 1189 UAUUUUAAAAAGUCACCAU 1229 AS1229-M5 CGAAAGGCUGC AUU S1158- CUGUCUUUGCCCAGAGCAUUGCAGC 1158 AAUGCUCUGGGCAAAGACA 1198 AS1198-M5 CGAAAGGCUGC GAG S1159- CUUGCCUGGAACUCACUCAUGCAGC 1159 AUGAGUGAGUUCCAGGCAA 1199 AS1199-M5 CGAAAGGCUGC GGA S1160- UUGCCUGGAACUCACUCACUGCAGC 1160 AGUGAGUGAGUUCCAGGCA 1200 AS1200-M5 CGAAAGGCUGC AGG S1161- AGAAUGACUUUUAUUGAGCUGCAGC 1161 AGCUCAAUAAAAGUCAUUC 1201 AS1201-M5 CGAAAGGCUGC UGC S1163- AUGACUUUUAUUGAGCUCUUGCAGC 1163 AAGAGCUCAAUAAAAGUCA 1203 AS1203-M5 CGAAAGGCUGC UUC S1184- GUAGCAUUUUUAUUAAUAUUGCAGC 1184 AAUAUUAAUAAAAAUGCUA 1224 AS1224-M5 CGAAAGGCUGC CAA S1185- AUUAAUAUGGUGACUUUUUAGCAGC 1185 UAAAAAGUCACCAUAUUAA 1225 AS1225-M5 CGAAAGGCUGC UAA S1186- UUAAUAUGGUGACUUUUUAAGCAGC 1186 UUAAAAAGUCACCAUAUUA 1226 AS1226-M6 CGAAAGGCUGC AUA S1186- UUAAUAUGGUGACUUUUUAAGCAGC 1186 UUAAAAAGUCACCAUAUUA 1226 AS1226-M5 CGAAAGGCUGC AUA S1190- AUGGUGACUUUUUAAAAUAAGCAGC 1190 UUAUUUUAAAAAGUCACCA 1230 AS1230-M5 CGAAAGGCUGC UAU S1191- UGGUGACUUUUUAAAAUAAAGCAGC 1191 UUUAUUUUAAAAAGUCACC 1231 AS1231-M5 CGAAAGGCUGC AUA S1192- GUGACUUUUUAAAAUAAAAAGCAGC 1192 UUUUUAUUUUAAAAAGUCA 1232 AS1232-M5 CGAAAGGCUGC CCA S1266- UGUUUUGCUUUUGUAACUU[U/A]G 1266 [U/A]AAGUUACAAAAGCA 1269 AS1269-M7 CAGCCGAAAGGCUGC AAACAGG S1266- UGUUUUGCUUUUGUAACUU[U/A]G 1266 [U/A]AAGUUACAAAAGCA 1269 AS1269-M8 CAGCCGAAAGGCUGC AAACAGG S1266- UGUUUUGCUUUUGUAACUU[U/A]G 1266 [U/A]AAGUUACAAAAGCA 1269 AS1269-M9 CAGCCGAAAGGCUGC AAACAGG S1267- UUUUGUAACUUGAAGAUAUAGCAGC 1267 UAUAUCUUCAAGUUACAAA 1270 AS1270-M10 CGAAAGGCUGC AGG S1268- CUGGGUUUUGUAGCAUUUUAGCAGC 1268 UAAAAUGCUACAAAACCCA 1271 AS1271-M11 CGAAAGGCUGC GGG S1268- CUGGGUUUUGUAGCAUUUUAGCAGC 1268 UAAAAUGCUACAAAACCCA 1271 AS1271-M9 CGAAAGGCUGC GGG S1266- UGUUUUGCUUUUGUAACUU[U/A]G 1266 [U/A]AAGUUACAAAAGCA 1269 AS1269-M12 CAGCCGAAAGGCUGC AAACAGG S1266- UGUUUUGCUUUUGUAACUU[U/A]G 1266 [U/A]AAGUUACAAAAGCA 1269 AS1269-M13 CAGCCGAAAGGCUGC AAACAGG

The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

It should be appreciated that, in some embodiments, sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and/or one or more modified nucleotides and/or one or more modified internucleotide linkages and/or one or more other modification compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 

1-50. (canceled)
 51. A method of treating a subject having or at risk of having hypercholesterolemia or atherosclerosis, the method comprising administering to the subject an oligonucleotide for reducing expression of PCSK9, the oligonucleotide comprising an antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 1219-1222, 1231-1232, and 1269-1271, and a sense strand comprising a sequence as set forth in any one of SEQ ID NOs: 1179-1182, 1191-1192, and 1266-1268, wherein the sense strand forms a duplex region with the antisense strand.
 52. The method of claim 51, wherein the antisense strand is up to 27 nucleotides in length.
 53. A method of treating a subject having or at risk of having hypercholesterolemia or atherosclerosis, the method comprising administering to the subject an oligonucleotide for reducing expression of PCSK9, wherein the oligonucleotide comprises a pair of antisense strand and sense strand, wherein the antisense strand is 21 to 27 nucleotides in length comprising a sequence as set forth in any one of SEQ ID NOs: 1219-1222, 1231-1232, and 1269-1271, and has a region of complementarity to PCSK9, wherein the sense strand comprises at its 3′-end a stem-loop set forth as: S₁-L-S₂, wherein S₁ is complementary to S₂, and wherein L forms a loop between S₁ and S₂ of 3 to 5 nucleotides in length, and wherein the antisense strand and the sense strand form a duplex structure of at least 19 nucleotides in length but are not covalently linked.
 54. The method of claim 53, wherein the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1179-1182, 1191-1192, and 1266-1268.
 55. The method of claim 51, wherein the oligonucleotide comprises a 3′-overhang sequence of two nucleotides in length, wherein the 3′-overhang sequence is present on the antisense strand.
 56. A method of treating a subject having or at risk of having hypercholesterolemia or atherosclerosis, the method comprising administering to the subject an oligonucleotide for reducing expression of PCSK9, wherein the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1219-1222, 1231-1232, and 1269-1271, and wherein the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1179-1182, 1191-1192, and 1266-1268; and wherein the oligonucleotide comprises at least one modified nucleotide, wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.
 57. The method of claim 56, wherein the oligonucleotide comprises at least one modified internucleotide linkage, and wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.
 58. The method of claim 56, wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.
 59. The method of claim 56, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, and wherein each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.
 60. The method of claim 53, wherein the oligonucleotide comprises at least one modified nucleotide, wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.
 61. The method of claim 53, wherein the oligonucleotide comprises at least one modified internucleotide linkage, and wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.
 62. The method of claim 53, wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.
 63. The method of claim 53, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, and wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.
 64. The method of claim 51, wherein the oligonucleotide comprises at least one modified nucleotide, wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.
 65. The method of claim 51, wherein the oligonucleotide comprises at least one modified internucleotide linkage, and wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.
 66. The method of claim 51, wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.
 67. The method of claim 51, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, and wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.
 68. The method of claim 51, wherein the subject has one or more symptoms or complications selected from the group consisting of coronary heart disease, angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke, feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, loss of consciousness, peripheral artery disease, and kidney problems.
 69. The method of claim 53, wherein the subject has one or more symptoms or complications selected from the group consisting of coronary heart disease, angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke, feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, loss of consciousness, peripheral artery disease, and kidney problems.
 70. The method of claim 56, wherein the subject has one or more symptoms or complications selected from the group consisting of coronary heart disease, angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke, feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, loss of consciousness, peripheral artery disease, and kidney problems. 